Selective proton coupled folate transporter and folate receptor, and garftase inhibitor compounds and methods of using the same

ABSTRACT

These compounds are useful in methods for treating cancer, selectively targeting cancerous cells via the proton coupled folate transporter, folate receptor alpha, and/or folate receptor beta pathways, inhibiting GARFTase in cancerous cells, and selectively targeting activated macrophages in a patient having an autoimmune disease, such as rheumatoid arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This utility patent application is a divisional patent application ofand claims the benefit of co-pending U.S. patent application Ser. No.16/000,954, Jun. 26, 2018, which is a divisional patent application ofU.S. patent application Ser. No. 15/342,359, filed Nov. 3, 2016, now U.SPat. No. 10,000,498, granted on Jun. 19, 2018, which is a divisionalpatent application of U.S. patent application Ser. No. 13/558,873, filedon Jul. 26, 2012, now U.S. Pat. No. 9,511,069, granted on Dec. 6, 2016,which is a divisional application of U.S. patent application Ser. No.12/242,988, filed Oct. 1, 2008, now U.S. Pat. No. 8,252,804, granted onAug. 28, 2012. The entire contents of U.S. patent application Ser. Nos.16/000,954, 15/342,359, 13/558,873 and 12/242,988, are incorporated byreference into this divisional utility patent application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No. R01CA125153 awaded by the National Institutes of Health, U.S. Department ofHealth and Human Services. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to selective proton coupled folatetransporter (PCFT) and alpha folate receptor (FR alpha), beta folatereceptor (FR beta), and glycinamide ribonucleotide formyltransferase(GARFTase) enzyme inhibitor compounds, and their methods of use.Preferably, these compounds have heterocycloalkyl-carbonyl-L-glutamatesubstituents or heterocycloaryl-carbonyl-L-glutamate substituents. Thecompounds of this invention may be made into salts that are watersoluble for providing orally active selective antitumor agents.

2. Description of the Background Art

Known cancer chemotherapy agents target both normal and cancerous tumorcells. This lack of selectivity for tumor cells results in cytotoxicityto the normal cells and is one of the major causes of chemotherapeuticfailure in the treatment of cancer. Further, advanced stage andchemotherapeutic agent resistant tumors may be difficult to treat withknow chemotherapeutic agents such as for example but not limited tocarboplatin or paclitaxel (docitaxel).

Folates are members of the B Class of vitamins that are cofactors forthe synthesis of nucleotide precursors. serine and methionine inone-carbon transfer reactions. Since mammals cannot synthesize folatesde novo, cellular uptake of these derivatives is essential for cellgrowth and tissue regeneration. Reflecting their hydrophilic anioniccharacter, folates do not cross biological membranes by diffusion alone.Accordingly, mammalian cells have evolved sophisticated membranetransport systems for facilitating accumulation of folates.

The ubiquitously expressed reduced folate carrier (RFC) is the majortransport system for folates in mammalian cells and mediatesconcentrative uptake of folate substrates. RFC is a member of the majorfacilitator superfamily of transporters and is an integral transmembraneprotein with micromolar affinity for its physiologic substrate, 5-methyltetrahydrofolate. Importantly, RFC is also the primary transporter ofclinically relevant antifolate drugs used for cancer includingmethotrexate (MTX), raltitrexed (ZD1694, Tomudex) (RTX), and pemetrexed(LY231514, Alimta) (PMX). Loss of RFC levels or function is a commonmode of antifolate resistance. While a previously unrecognizedproton-coupled folate transporter (PCFT) was recently reported tocontribute to folate absorption in the duodenum, its tissue-specificityand overall role in folate homeostasis are not clear yet.

The family of folate receptors (FRs) represents yet another mode offolate uptake into mammalian cells. The FRs are high affinity folatebinding proteins encoded by three distinct genes, designated FR alpha,FR beta and FR gamma, localized to chromosome 11q13.3-q13.5. In contrastto RFC and PCFT, FR alpha and FR beta are anchored in plasma membranesby a glycosyl phosphatidylinositol (GPI) anchor. FR gamma contains noGPI anchor and is secreted. Whereas FR alpha and FR beta (but not FRgamma) mediate cellular accumulation of folate at low (nanomolar)concentrations by receptor-mediated endocytosis, these homologousproteins show differences in binding affinities for reduced folatesubstrates.

The high affinity FRs offer a potential means of selective tumortargeting, given their restricted pattern of tissue expression andfunction. For instance, FR alpha is expressed on the apical membranesurface of normal tissues such as kidney, placenta, and choroid plexus,whereas FR beta is expressed in placenta, spleen, and thymus.Importantly, FR alpha is overexpressed in a number of carcinomasincluding up to 90% of ovarian cancers. Close associations were reportedbetween FR alpha expression levels with grade and differentiation statusof ovarian tumors. FR alpha in normal tissues (unlike tumors) isreported to be inaccessible to the circulation. FR beta is expressed ina wide range of myeloid leukemia cells. FR beta in normal hematopoeticcells differs from that in leukemia cells in its inability to bindfolate ligand.

Folate-conjugated cytotoxins, liposomes, or radionuclides, or cytoxicantifolates have all been used to target FRs. Unfortunately, for mostfolate-based therapeutics such as classical antifolates (including RTX,PMX, and lometrexol (LMX)), tumor selectivity is lost since substratesare shared between FRs and the ubiquitously expressed RFC. Indeed, thislikely explains the severe myelosupression encountered in phase 1studies with LMX.

If, a FR-targeted ligand were itself cytotoxic without RFC activity,selective tumor targeting would ensue. Antifolates that selectivelytarget FRs over RFC have been described including CB3717 and, morerecently, cyclopenta[g]quinazoline antifolates BGC638 and BGC945, all ofwhich potently inhibit thymidylate synthase (TS) within cells. WhenBGC945 was tested in mice, there was no toxicity to normal tissues, asreflected in weight loss, nor were there any macroscopic signs oftoxicity to major organs, consistent with the premise that FR targetingis highly selective.

As is known by those skilled in the art, FRs such as FR alpha and FRbeta are overexpressed on a substantial amount of certain surfaces of anumber of types of cancerous tumors. FR alpha is known to beoverexpressed in ovarian, endometrial, kidney, lung, mesothelioma,breast and brain tumors. FR beta is known to be overexpressed in acutemyeloid leukemias

In most normal cells, the FRs are not present. In most normal cells,folic acid is not taken up by way of a reduced folate carrier (RFC)system. Uptake of folates and antifolates by tissues and tumors isprimarily by the ubiquitously expressed RFC system. In light of thespecificity of folic acid, conjugates of folic acid have been used toselectively deliver toxins, liposomes, imaging agents, and cytotoxicagents to FR expressing tumors. The major limitation of the folic acidconjugates is that they require cleavage from the folic acid moiety torelease, for example, the cytotoxic agent. Cleavage of the cytotoxicagent moiety from the folic acid conjugate is often difficult to achieveand the anti-tumor activity is hindered or is nonexistent as a result ofthe inability or reduced ability to release the cytotoxic agent. Anotherlimitation of the folic acid conjugates entails premature release of thecytotoxic agent during transport and before reaching the canceroustumor. The premature release thus leads to undesired toxicity in normalcells.

The FRs alpha and beta represent another mode of folate uptake and areconsidered by those skilled in the art to be potential chemotherapeutictargets for selective tumor uptake. US Patent Application PublicationNo. US 2008/0045710 A1, published Feb. 21, 2008 (Aleem Gangjee)describes compounds for treating cancer tumors wherein fused cyclicpyrimidines are used to selectively target FRs of cancerous tumors thatexpress FR alpha and FR beta and that inhibit glycinamide ribonucleotideformyltransferase (GARFTase) enzyme. The compounds are not significantlytaken up by a cell or tissue using the RFC system.

There is a need for single compounds having potent anti-tumor activitythat selectively target FR alpha and FR beta of cancerous cells, thatinhibit GARFTase in cancerous cells, and that have a negligiblesubstrate activity for RFC.

SUMMARY OF THE INVENTION

The present invention meets the above need by providing selective protoncoupled folate transporter (PCFT) and alpha and beta FR, and GARTFaseenzyme inhibitor compounds.

The present invention provides a compound comprising Formula I:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d); R₂ comprises one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A comprises one of(a) CR′R″, (b) NR′, wherein R′ and R″ are the same or different and areeither a H or an alkyl group having from 1 to 6 carbon atoms, (c) asulfur (S), and (d) an oxygen (O); wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and wherein when said sidechain attachment is at position 7 then A comprises one of (a) CR′, and(b) N, and optionally includes wherein the carbon atoms at positions 5and 6, independently, have attached thereto either (a) two hydrogenatoms if the bond between carbon atoms 5 and 6 is a single bond or onehydrogen atom if the bond between carbon atoms 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms and a hydrogenatom if the bond between carbon atoms at positions 5 and 6 is a singlebond or an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms 5 and 6 is a double bond, and combinations thereof;and R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, wherein the sum total of integers yand z is equal to or less than seven.

Another embodiment of this invention comprises the compound of FormulaI, as described herein, wherein the side chain attachment is at carbonatom position 6 and wherein A is CR′R″, and wherein the carbon atom atposition 5, independently has attached thereto either (a) two hydrogenatoms if the bond between carbon atoms at positions 5 and 6 is a singlebond or one hydrogen atom if the bond between carbon atoms at positions5 and 6 is a double bond, or (b) an alkyl group having from one to sixcarbon atoms if the bond between carbon atoms of positions 5 and 6 is adouble bond or an alkyl group having from one to six carbon atoms and ahydrogen atom if the bond between carbon atoms at positions 5 and 6 is asingle bond, and combinations thereof.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, is provided comprising wherein the side chainattachment is at carbon atom position 6 and wherein A is NR′ wherein R′is either a hydrogen atom or an alkyl group having from one to sixcarbon atoms, and wherein the carbon atom at position 5, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

In yet another embodiment of this invention, a compound of Formula I, asdescribed herein, is provided comprising wherein said side chainattachment is at carbon atom position 5 and wherein and wherein A isCR′R″, and wherein the carbon atom at position 6, independently hasattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

Another embodiment of this invention provides a compound of Formula I,as described herein, comprising wherein the side chain attachment is atcarbon atom position 5 and wherein A is NR′ wherein R′ is either ahydrogen atom or an alkyl group having from one to six carbon atoms, andwherein the carbon atom at position 6, independently has attachedthereto either (a) two hydrogen atoms if the bond between carbon atomsat positions 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a double bond, or (b) analkyl group having from one to six carbon atoms if the bond betweencarbon atoms of positions 5 and 6 is a double bond or an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, comprises the side chain having one or more carbon tocarbon double or triple bonds between the carbon atoms of (C) _(y) and(C) z .

In a preferred embodiment of this invention, the compound of Formula I,as described herein, is provided comprising wherein A is NR′ and R′ is ahydrogen atom, and wherein y is from one to six carbon atoms, z is zero,R₃, and R₅ are each hydrogen atoms, and X is selected from the groupconsisting of a heterocycloalkyl-carbonyl-L-glutamate group and aheterocycloaryl-carbonyl-L-glutamate group. Theheterocycloalkyl-carbonyl-L-glutamate group is selected from the groupconsisting of a dihydrothiophene-carbonyl-L-glutamate group, atetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof. Theheterocycloaryl-carbonyl-L-glutamate group is selected from the groupconsisting of a thiophene-carbonyl-L-glutamate group, afuran-carbonyl-L-glutamate group, a pyrrole-carbonyl-L-glutamate group,and a pyridine-carbonyl-L-glutamate group.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, provides wherein the side chain of Formula I compriseszero or one or more double bonds comprising E-isomers and Z-isomers.

Other embodiments of this invention provide for the R and S opticalisomers of the heterocyclic compounds of the present invention when thedouble bond of the ring system is broken.

Other embodiments of this invention provide a pharmaceutical compositionhaving a therapeutically effective amount of a compound comprisingFormula I, and a pharmaceutically acceptable salt, prodrug, solvate orhydrate of the compound comprising Formula I, as described herein.

Further embodiments of this invention provide methods for treatingcancer, targeting cancerous cells via the proton coupled folatetransporter pathway, inhibiting GARFTase in cancerous cells, andselectively targeting activated macrophages in a patient having anautoimmune disease, such as rheumatoid arthritis.

A preferred embodiment of the present invention provides for a compoundcomprising Formula II:

wherein R₁ comprises one of a hydrogen (H) or an alkyl group having from1 to 6 carbon atoms;

R₂ comprises one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms;

A comprises one of (a) CR′R″, (b) NR′, wherein R′ and R″ are the same ordifferent and are either a H or an alkyl group having from 1 to 6 carbonatoms, (c) a sulfur (S), and (d) an oxygen (O);

wherein the bond at position 5-6 is a double bond;

wherein the five membered ring has a side chain attached at position 6,and optionally includes wherein the carbon atoms at positions 5 and 6,independently, have attached thereto either (a) one hydrogen atom, or(b) an alkyl group having from one to six carbon atoms, and combinationsthereof; and

R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl,(d) difluoromethyl, (e) monofluoromethyl, (f) methyl ketone, (g)trifluoromethyl ketone, (h) difluoromethyl ketone, (i) monofluoromethylketone, (j) formyl, (k) methyl alcohol, (l) methylamine, or (m) a bond;

B is one of (a) a sulfur (S) atom, (b) an oxygen (O) atom, or (c) anitrogen (N) atom; and

y is an integer ranging from zero up to and including 7.

Another embodiment of this invention provides the compound of Formula IIcomprising wherein the side chain has one or more carbon to carbondouble or triple bonds between the carbon atoms of (C)_(y 1-7). Inanother embodiment of this invention the compound of Formula IIcomprises wherein the side chain comprises zero or one or more doublebonds comprising E-isomers and Z-isomers. Another embodiment providesthe compound of Formula II comprising one of a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate thereof. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula II is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention may be gained from the followingdescription of the preferred embodiments of the when read in conjunctionwith the accompanying drawings in which:

FIG. 1 shows the chemical structures of seven compounds of the presentinvention, namely, sample IDs AAG154353, AAG154360, AAG154484,AAG154468, AAG154479, AAG154489, AAG154485, and AAG154544.

FIG. 2 shows the biological effects of various compounds of the presentinvention.

FIG. 3 shows the structure of a comparison compound AAG120366-2 .

FIG. 4 shows hRFC and hPCFT and inhibition by Compounds AAG120366-2(shown as “Q” in FIG. 6), AAG154353 (shown as “71” in FIG. 6), and PT523(shown as “PT” in FIG. 6). Assays used ³H-methotrexate at 37° C. in pH5.5 IVIES-buffered saline without additions (shown as “NA” in FIG. 6) orin the presence of 1 or 10 micromolar inhibitor Compounds “Q”, “71”, and“PT” as set forth in FIG. 6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a compound comprising Formula I:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d); R₂ comprises one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A comprises one of(a) CR′R″, (b) NR′, wherein R′ and R″ are the same or different and areeither a H or an alkyl group having from 1 to 6 carbon atoms, (c) asulfur (S), and (d) an oxygen (O); wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and wherein when said sidechain attachment is at position 7 then A comprises one of (a) CR′, and(b) N, and optionally includes wherein the carbon atoms at positions 5and 6, independently, have attached thereto either (a) two hydrogenatoms if the bond between carbon atoms 5 and 6 is a single bond or onehydrogen atom if the bond between carbon atoms 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms and a hydrogenatom if the bond between carbon atoms at positions 5 and 6 is a singlebond or an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms 5 and 6 is a double bond, and combinations thereof;and R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, wherein the sum total of integers yand z is equal to or less than seven.

Another embodiment of this invention comprises the compound of FormulaI, as described herein, wherein the side chain attachment is at carbonatom position 6 and wherein A is the CR′R″, and wherein the carbon atomat position 5, independently has attached thereto either (a) twohydrogen atoms if the bond between carbon atoms at positions 5 and 6 isa single bond or one hydrogen atom if the bond between carbon atoms atpositions 5 and 6 is a double bond, or (b) an alkyl group having fromone to six carbon atoms if the bond between carbon atoms of positions 5and 6 is a double bond or an alkyl group having from one to six carbonatoms and a hydrogen atom if the bond between carbon atoms at positions5 and 6 is a single bond, and combinations thereof.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, is provided comprising wherein the side chainattachment is at carbon atom position 6 and wherein A is NR′ wherein R′is either a hydrogen atom or an alkyl group having from one to sixcarbon atoms, and wherein the carbon atom at position 5, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

In yet another embodiment of this invention, a compound of Formula I, asdescribed herein, is provided comprising wherein said side chainattachment is at carbon atom position 5 and wherein and wherein A is theCR′R″, and wherein the carbon atom at position 6, independently hasattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

Another embodiment of this invention provides a compound of Formula I,as described herein, comprising wherein the side chain attachment is atcarbon atom position 5 and wherein A is NR′ wherein R′ is either ahydrogen atom or an alkyl group having from one to six carbon atoms, andwherein the carbon atom at position 6, independently has attachedthereto either (a) two hydrogen atoms if the bond between carbon atomsat positions 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a double bond, or (b) analkyl group having from one to six carbon atoms if the bond betweencarbon atoms of positions 5 and 6 is a double bond or an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

The heterocycloalkyl-carbonyl-L-glutamate group is selected from thegroup consisting of a dihydrothiophene-carbonyl-L-glutamate group, atetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof.

The heterocycloaryl-carbonyl-L-glutamate group is selected from thegroup consisting of a thiophene-carbonyl-L-glutamate group, afuran-carbonyl-L-glutamate group, a pyrrole-carbonyl-L-glutamate group,and a pyridine-carbonyl-L-glutamate group.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, comprises the side chain having one or more carbon tocarbon double or triple bonds between the carbon atoms of (C)_(y) and(C)_(z).

In a preferred embodiment of this invention, the compound of Formula I,as described herein, is provided comprising wherein A is NR′ and R′ is ahydrogen atom, and wherein y is from one to six carbon atoms , z iszero, R₃, and R₅ are each hydrogen atoms, and X is selected from thegroup consisting of a heterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group.

In another embodiment of this invention, the compound of Formula I, asdescribed herein, provides wherein the side chain of Formula I compriseszero or one or more double bonds comprising E-isomers and Z-isomers.

Another embodiment of this invention provides a pharmaceuticallyacceptable salt, prodrug, solvate or hydrate of the compound of FormulaI, as described herein.

In yet another embodiment of this invention, a pharmaceuticalcomposition is provided comprising a therapeutically effective amount ofa compound comprising Formula I:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d); R₂ comprises one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A comprises one of(a) CR′R″, (b) NR′, wherein R′ and R″ are the same or different and areeither a H or an alkyl group having from 1 to 6 carbon atoms, (c) asulfur (S), and (d) an oxygen (O); wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and wherein when said sidechain attachment is at position 7 then A comprises one of (a) CR′, and(b) N, and optionally includes wherein the carbon atoms at positions 5and 6, independently, have attached thereto either (a) two hydrogenatoms if the bond between carbon atoms 5 and 6 is a single bond or onehydrogen atom if the bond between carbon atoms 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms and a hydrogenatom if the bond between carbon atoms at positions 5 and 6 is a singlebond or an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms 5 and 6 is a double bond, and combinations thereof;and R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, wherein the sum total of integers yand z is equal to or less than seven.

In another embodiment of this invention, the pharmaceutical compositioncomprises wherein the side chain attachment is at carbon atom position 6and wherein A is CR′R″, and further comprising wherein the carbon atomat position 5, independently has attached thereto either (a) twohydrogen atoms if the bond between carbon atoms at positions 5 and 6 isa single bond or one hydrogen atom if the bond between carbon atoms atpositions 5 and 6 is a double bond, or (b) an alkyl group having fromone to six carbon atoms if the bond between carbon atoms of positions 5and 6 is a double bond or an alkyl group having from one to six carbonatoms and a hydrogen atom if the bond between carbon atoms at positions5 and 6 is a single bond, and combinations thereof.

In another embodiment of this invention, the pharmaceutical compositionof Formula I comprises wherein the side chain attachment is at carbonatom position 6 and wherein A is NR′ wherein R′ is either a hydrogenatom or an alkyl group having from one to six carbon atoms, and whereinthe carbon atom at position 5, independently has attached thereto either(a) two hydrogen atoms if the bond between carbon atoms at positions 5and 6 is a single bond or one hydrogen atom if the bond between carbonatoms at positions 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms if the bond between carbon atoms ofpositions 5 and 6 is a double bond or an alkyl group having from one tosix carbon atoms and a hydrogen atom if the bond between carbon atoms atpositions 5 and 6 is a single bond, and combinations thereof.

Another embodiment of this invention provides the pharmaceuticalcomposition of Formula I comprising wherein the side chain attachment isat carbon atom position 5 and wherein and wherein A is CR′R″, andfurther comprising wherein the carbon atom at position 6, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

A further embodiment of this invention provides the pharmaceuticalcomposition of Formula I comprising wherein the side chain attachment isat carbon atom position 5 and wherein A is NR′ wherein R′ is either ahydrogen atom or an alkyl group having from one to six carbon atoms, andwherein the carbon atom at position 6, independently has attachedthereto either (a) two hydrogen atoms if the bond between carbon atomsat positions 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a double bond, or (b) analkyl group having from one to six carbon atoms if the bond betweencarbon atoms of positions 5 and 6 is a double bond or an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

Another embodiment of this invention provides the pharmaceuticalcomposition of Formula I comprising the side chain having one or morecarbon to carbon double or triple bonds between the carbon atoms of (C)_(y) and (C) z .

In a preferred embodiment of this invention, the pharmaceuticalcomposition of Formula I comprises wherein A is NR′ and R′ is a hydrogenatom, and wherein y is from one to six carbon atoms , z is zero, R₃, andR₅ are each hydrogen atoms, and X is selected from the group consistingof a heterocycloalkyl-carbonyl-L-glutamate group and aheterocycloaryl-carbonyl-L-glutamate group.

In another embodiment of this invention, the pharmaceutical compositionof Formula I comprises wherein said the side chain of Formula Icomprises zero or one or more double bonds comprising E-isomers andZ-isomers.

This invention provides for a pharmaceutically acceptable salt, prodrug,solvate or hydrate of the pharmaceutical composition of Formula I, asdescribed herein.

A method of treating a patient diagnosed with cancer is provided in thisinvention comprising administering to a patient a therapeuticallyeffective amount of a compound of Formula I:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d); R₂ comprises one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A comprises one of(a) CR′R″, (b) NR′, wherein R′ and R″ are the same or different and areeither a H or an alkyl group having from 1 to 6 carbon atoms, (c) asulfur (S), and (d) an oxygen (O); wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and wherein when said sidechain attachment is at position 7 then A comprises one of (a) CR′, and(b) N, and optionally includes wherein the carbon atoms at positions 5and 6, independently, have attached thereto either (a) two hydrogenatoms if the bond between carbon atoms 5 and 6 is a single bond or onehydrogen atom if the bond between carbon atoms 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms and a hydrogenatom if the bond between carbon atoms at positions 5 and 6 is a singlebond or an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms 5 and 6 is a double bond, and combinations thereof;and R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; and z is an integerranging from zero up to and including seven, wherein the sum total ofintegers y and z is equal to or less than seven.

In another embodiment of this invention, the method of treating apatient with cancer, as described herein, includes administering to thepatient a compound of Formula I comprising wherein the side chainattachment is at carbon atom position 6 and wherein A is CR′R″, andfurther comprising wherein the carbon atom at position 5, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

Another embodiment of this invention provides a method of treating apatient with cancer, as described herein, including administering to thepatient a compound of Formula I comprising wherein the side chainattachment is at carbon atom position 6 and wherein A is NR′ wherein R′is either a hydrogen atom or an alkyl group having from one to sixcarbon atoms, and wherein the carbon atom at position 5, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.

In another embodiment of this invention, a method of treating a patientwith cancer, as described herein, includes administering to the patienta compound of Formula I wherein the side chain attachment is at carbonatom position 5 and wherein A is CR′R″, and further comprising whereinthe carbon atom at position 6, independently has attached thereto either(a) two hydrogen atoms if the bond between carbon atoms at positions 5and 6 is a single bond or one hydrogen atom if the bond between carbonatoms at positions 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms if the bond between carbon atoms ofpositions 5 and 6 is a double bond or an alkyl group having from one tosix carbon atoms and a hydrogen atom if the bond between carbon atoms atpositions 5 and 6 is a single bond, and combinations thereof.

In another embodiment of this invention, a method of treating a patientwith cancer, as described herein, includes administering to a patient acompound of Formula I wherein the side chain attachment is at carbonatom position 5 and wherein A is NR′ wherein R′ is either a hydrogenatom or an alkyl group having from one to six carbon atoms, and whereinthe carbon atom at position 6, independently has attached thereto either(a) two hydrogen atoms if the bond between carbon atoms at positions 5and 6 is a single bond or one hydrogen atom if the bond between carbonatoms at positions 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms if the bond between carbon atoms ofpositions 5 and 6 is a double bond or an alkyl group having from one tosix carbon atoms and a hydrogen atom if the bond between carbon atoms atpositions 5 and 6 is a single bond, and combinations thereof.

The methods of treating a patient with cancer, as described herein,include wherein the heterocycloalkyl-carbonyl-L-glutamate group isselected from the group consisting of adihydrothiophene-carbonyl-L-glutamate group, atetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof, andwherein the heterocycloaryl-carbonyl-L-glutamate group is selected fromthe group consisting of a thiophene-carbonyl-L-glutamate group, afuran-carbonyl-L-glutamate group, a pyrrole-carbonyl-L-glutamate group,and a pyridine-carbonyl-L-glutamate group.

The methods of treating a patient with cancer, as described herein,include administering to the patient an effective amount of the compoundof Formula I wherein the side chain has one or more carbon to carbondouble or triple bonds between the carbon atoms of (C)_(y) and (C)_(z).

Preferably, the method of treating a patient with cancer, as describedherein, includes administering to the patient an effective amount of thecompound of Formula I wherin A is NR′ and R′ is a hydrogen atom, andwherein y is from one to six carbon atoms, z is zero, R₃, and R₅ areeach hydrogen atoms, and X is selected from the group consisting of aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, as described herein. Themethod of treating a patient with cancer, as described herein, includesadministering to the patient an effective amount of a compound ofFormula I wherein the side chain of Formula I comprises zero or one ormore double bonds comprising E-isomers and Z-isomers.

All of the methods of treating a patient with cancer, as describedherein, include administering to the patient an effective amount of theCompound of Formula I, as described herein, or a pharmaceuticallyacceptable salt, prodrug, solvate or hydrate of the compound of FormulaI, as described herein.

A method for targeting cancerous cells via the proton coupled folatetransporter pathway is provided comprising:

(a) providing a compound comprising Formula I:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d);

R₂ comprises one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms;

A comprises one of (a) CR′R″, (b) CH₃, wherein R′ and R″ are the same ordifferent and are either a H or an alkyl group having from 1 to 6 carbonatoms, (c) a sulfur (S), and (d) an oxygen (O);

wherein the bond at position 5-6 may either be a single or a doublebond;

wherein the five membered ring has a side chain attached at positions 5,6 or 7, and wherein when said side chain attachment is at position 7then A comprises one of (a) CR′, and (b) N, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof, and

R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl,(d) difluoromethyl, (e) monofluoromethyl, (f) methyl ketone, (g)trifluoromethyl ketone, (h) difluoromethyl ketone, (i) monofluoromethylketone, (j) formyl, (k) methyl alcohol, (l) methylamine, or (m) a bond;

X is either a heterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond;

wherein R₅ is the same as R₃ except that R₅ is not a bond;

y is an integer ranging from zero up to and including 6;

z is an integer ranging from zero up to and including seven, wherein thesum total of integers y and z is equal to or less than seven;

(b) subjecting cancerous cells expressing a human proton coupled folatetransporter (PCFT) to said compound of Formula I;

(c) establishing selective binding of said compound of Formula Ito saidhuman PCFT; and

(d) effecting the selective transport of said compound of Formula Ibound to said human PCFT to a target cancerous cell wherein saidcompound of Formula I acts as a growth inhibitor of said targetcancerous cells and inhibits GARFTase within said target cancerouscells.

Another embodiment of this method for targeting cancerous cells of thisinvention, as described herein, include wherein the compound of FormulaI is selective for receptors of FR alpha and human PCFT associated withexpressing cancerous cells. In this method of targeting cancerous cell,the compound of Formula I is not significantly taken up by tissues orcells using the reduced folate carrier (RFC) system.

Other embodiments of this method for targeting cancerous cells compriseemploying any of the various compounds of Formula I, or apharmaceutically acceptable salt, prodrug, solvate or hydrate of acompound of Formula I, as described herein, thus it will be understoodby those skilled in the art that any of the positions for attaching theside chain, as described herein, are embodiments of this invention.These methods for targeting cancer cells include wherein the compoundtargets cancerous cells selected from the group consisting of ovarian,breast, cervical, and kidney brain tumors.

A method for inhibiting GARFTase in cancerous cells is providedcomprising:

(a) providing a compound of Formula I having a cytotoxic affect:

wherein R₁ comprises one of (a) a hydrogen (H)), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d);

R₂ comprises one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms;

A comprises one of (a) CR′R″, (b) NR′, wherein R′ and R″ are the same ordifferent and are either a H or an alkyl group having from 1 to 6 carbonatoms, (c) a sulfur (S), and (d) an oxygen (O);

wherein the bond at position 5-6 may either be a single or a doublebond;

wherein the five membered ring has a side chain attached at positions 5,6 or 7, and wherein when said side chain attachment is at position 7then A comprises one of (a) CR′, and (b) N, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof, and

R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl,(d) difluoromethyl, (e) monofluoromethyl, (f) methyl ketone, (g)trifluoromethyl ketone, (h) difluoromethyl ketone, (i) monofluoromethylketone, (j) formyl, (k) methyl alcohol, (l) methylamine, or (m) a bond;

X is either a heterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond;

wherein R₅ is the same as R₃ except that R₅ is not a bond;

y is an integer ranging from zero up to and including 6;

z is an integer ranging from zero up to and including seven, wherein thesum total of integers y and z is equal to or less than seven;

(b) selectively delivering said compound to said cancerous cell;

(c) effecting the entry of said compound into said cancerous cell;

(d) retaining said compound in said cancerous cell for a sufficientamount of time for effecting binding of said compound with a GARFTaseenzyme; and

(e) lysing of said cancerous cell via said binding of said compound withsaid GARFTase enzyme and inhibiting the DNA replication of saidcancerous cell.

Preferably, the method, of this invention, of inhibiting GARFTase, asdescribed herein, comprises wherein the compound of Formula I or apharmaceutically acceptable salt, prodrug, solvate or hydrate of thecompound of Formula I is selective for receptors of FR alpha associatedwith expressing cancerous cells.

Other embodiments of this invention of inhibiting GARTase, as describedherein, include employing any one of the various embodiments of thecompound of Formula I or its pharmaceutically acceptable salt, prodrug,solvate or hydrate, as described herein, including comprising the sidechain attachment at various positions 5, 6 or 7, as described herein.

Another embodiment of this invention provides for the inhibition ofAICARFTase when A is equal to a sulfur atom in the compound of FormulaI.

Rheumatoid arthritis is an autoimmune disease that affects the qualityof life of millions of patients worldwide. Rheumatoid arthritis ischaracterized by inflammation of a patient's joints and destruction ofthe cartilage and bone of the patient. While the pathology of rheumatoidarthritis is complex, it is known to involve the infiltration andactivation of immune cells along with the release of destructiveinflammatory mediators into a patient's synovium of affected joints.Paulos, Chrystal M., et al., “Folate receptor-mediated targeting oftherapeutic and imaging agents to activated macrophages in rheumatoidarthritis”, Advanced Drug Delivery Reviews, Vol. 56, pages 1205-1217(2004), describe the discovery of folate receptor expression onactivated macrophage cells in patient models (human and animal) withnaturally occurring rheumatoid arthritis, and is incorporated herein byreference, specifically section 3, page 1208 and section 5, pages1212-1214.

The present invention provides a method for selectively targetingactivated macrophages in a patient having an autoimmune diseasecomprising:

(a) providing a compound comprising Formula I:

wherein R₁ comprises one of (a) a hydrogen (H), (b) an OH, (c) CH₃, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms, and tautomers of (b) and (d);

R₂ comprises one of (a) a hydrogen (H), (b) a CH₃,(c) an OH, and (d) NHRwherein R is either a H or an alkyl group having from 1 to 6 carbonatoms;

A comprises one of (a) CR′R″, (b) NR′, wherein R′ and R″ are the same ordifferent and are either a H or an alkyl group having from 1 to 6 carbonatoms, (c) a sulfur (S), and (d) an oxygen (O);

wherein the bond at position 5-6 may either be a single or a doublebond;

wherein the five membered ring has a side chain attached at positions 5,6 or 7, and wherein when said side chain attachment is at position 7then A comprises one of (a) CR′, and (b) N, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof, and

R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl,(d) difluoromethyl, (e) monofluoromethyl, (f) methyl ketone, (g)trifluoromethyl ketone, (h) difluoromethyl ketone, (i) monofluoromethylketone, (j) formyl, (k) methyl alcohol, (l) methylamine, or (m) a bond;

X is either a heterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group , and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond;

wherein R₅ is the same as R₃ except that R₅ is not a bond;

y is an integer ranging from zero up to and including 6;

z is an integer ranging from zero up to and including seven, wherein thesum total of integers y and z is equal to or less than seven;

(b) subjecting an activated macrophage expressing a folate receptor (FR)to said compound of Formula I;

(c) establishing selective binding of said compound of Formula I to saidFR; and

(d) effecting the selective transport of said compound of Formula Ibound to said FR to a target activated macrophage of the autoimmunedisease wherein said compound of Formula I acts as an inhibitor of saidactivated macrophage's release of destructive inflammatory mediators.

The method for selectively targeting activated macrophages of thepresent invention includes wherein the compound of Formula I isselective for receptors of FR alpha and human proton coupled folatetransporter (PCFT) associated with expressing macrophage cells.

Preferably, the method for selectively targeting activated macrophagesin a patient having an autoimmune disease, as described herein, includeswherein the activated macrophage cell expressing the FR is rheumatoidarthritis.

Other embodiments of the method for targeting activated macrophage cellsin a patient with an autoimmune disease, include wherein the compound ofFormula I, or its pharmaceutically acceptable salts, prodrugs, solvatesor hydrates of the compound of Formula I, include any of the variousembodiments, as described herein, of the compound of Formula I,including attachment of the side chain at any of the positions 5, 6, or7, as described herein.

Preferably, the method of selectively targeting an activated macrophagein a patient having an autoimmune disease that is rheumatoid arthritisincludes delivering the compound of Formula I or a pharmaceuticallyacceptable salt, prodrug, solvate or hydrate of the compound of FormulaI by injection into a joint or synovial fluid of a patient.

A preferred embodiment of the present invention provides for a compoundcomprising Formula II:

wherein R₁ comprises one of a hydrogen (H) or an alkyl group having from1 to 6 carbon atoms;

R₂ comprises one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms;

A comprises one of (a) CR′R″, (b) CH₃, wherein R′ and R″ are the same ordifferent and are either a H or an alkyl group having from 1 to 6 carbonatoms, (c) a sulfur (S), and (d) an oxygen (O);

wherein the bond at position 5-6 is a double bond;

wherein the five membered ring has a side chain attached at position 6,and optionally includes wherein the carbon atoms at positions 5 and 6,independently, have attached thereto either (a) one hydrogen atom, or(b) an alkyl group having from one to six carbon atoms, and combinationsthereof; and

R₃ comprises one of (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl,(d) difluoromethyl, (e) monofluoromethyl, (f) methyl ketone, (g)trifluoromethyl ketone, (h) difluoromethyl ketone, (i) monofluoromethylketone, (j) formyl, (k) methyl alcohol, (l) methylamine, or (m) a bond;

B is one of (a) a sulfur (S) atom, (b) an oxygen (O) atom, or (c) anitrogen (N) atom; and

y is an integer ranging from zero up to and including 7.

Another embodiment of this invention provides the compound of Formula IIcomprising wherein the side chain has one or more carbon to carbondouble or triple bonds between the carbon atoms of (C)_(y 1-7). Inanother embodiment of this invention the compound of Formula IIcomprises wherein the side chain comprises zero or one or more doublebonds comprising E-isomers and Z-isomers. Another embodiment providesthe compound of Formula II comprising one of a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate thereof. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula II is also provided. Other preferred embodiments of thepresent invention provide for methods as described herein for treatingcancer, selectively targeting cancerous cells via the proton coupledfolate transporter, folate receptor alpha, and/or folate receptor betapathways, inhibiting GARFTase in cancerous cells, and selectivelytargeting activated macrophages in a patient having an autoimmunedisease employing the compound of Formula II as the preferred tautomerprovided by the compound of Formula I.

While the Formula II shows attachments of the five membered ring of theside chain to be at the 2 and 5 positions (numbering clockwise with “B”being at position 1), the substituents attached to the five memberedring of the side chain may be at various positions, including forexample, at the 2 and 3 positions, at the 2 and 4 positions, at the 3and 4 positions, and at the 3 and 5 positions.

As used herein, the term “patient” means members of the animal kingdom,including, but not limited to, human beings. As used herein, the term“having cancer” means that the patient has been diagnosed with cancer.

As used herein, the term “therapeutically effective amount” refers tothat amount of any of the present compounds required to bring about adesired effect in a patient. The desired effect will vary depending onthe illness being treated. For example, the desired effect may bereducing tumor size, destroying cancerous cells, and/or preventingmetastasis, any one of which may be the desired therapeutic response. Onits most basic level, a therapeutically effective amount is that amountneeded to inhibit the mitosis of a cancerous cell.

Compounds of the present invention covered under Formula I or II, andpharmaceutically acceptable salts, prodrugs, solvates or hydratesthereof, may also be administered with one or more additional treatmentagents, i.e., a chemotherapeutic agent. Suitable candidates for theadditional chemotherapeutic agent include for example but are notlimited to, paclitaxel, docetaxel, vinca alkaloids, colchicines,colcemid, cisplatin, and nocadazol.

As used herein, the term “lower alkyl” group refers to those lower alkylgroups having one to about ten carbon atoms, such as for example methyl,ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,cyclohexyl, cyclopropylmethyl or cyclobutylmethyl groups. Alkyl groupssharing one to about six carbon atoms are preferred. These lower alkylgroups are straight chain, branched chain or cyclic (alicyclichydrocarbon) arrangements. The carbon atoms of these straight chain,branched chain or cyclic arranged alkyl groups may have one or moresubstituents for the hydrogens attached to the carbon atoms.

As used herein, the term “heteroalkyl” refers to alkyl chains from oneto about 3 atoms where one or more of the carbons has been replaced withnitrogen, oxygen or sulfur, Thus “heteroalkyl” groups will include, forexample, C—C—N, C—S, S—C, C—C—O, O—C, N—C—C, N—C═C and other variouscombinations, as will be apparent to one skilled in the art. The abovelist is not meant to be exhaustive, and many combinations arecontemplated as within the scope of the present invention.

The term “aryl” groups, as used herein, refers to compounds whosemolecules have an aromatic ring structure, such as the six-carbon ringof benzene, or multiple rings which are either fused or unfused, such ascondensed six-carbon rings of other aromatic derivatives. The term“aryl” is also defined to include diaryl, triaryl and polyaryl groups,which would have two, three or more rings, respectively. Thus, suitablearyl groups would include, for example, phenyl, biphenyl, naphthyl,phenanthrene, anthracene groups and aryl oxyaryl groups. This list isnot meant to be exhaustive, and any aryl group, as these terms aredefined above and commonly understood in the art, are within the scopeof the present invention.

The term “heteroaryl” refers to aromatic ring structures having at leastone atom in the ring which is not carbon, such as oxygen, nitrogen orsulfur. “Heteroaryls” as used herein also refers to aromatic ringstructures that are part of larger ring structures, such as two or threemember ring systems, which may be fused or unfused, in which one of therings is as described above. Thus, “heteroaryl” refers to ring systemsin which one or more rings contain a heteroatom and one or more rings donot. It will be understood that this list is not meant to be exhaustive,and that any heteroaryl group, as these terms are defined above andcommonly understood in the art, are within the scope of the presentinvention. The heteroaryl ring systems may be fused ring systems orunfused. Examples of heteroaryl ring systems include, for example butare not limited to, pyridine, quinoline, isoquinoloine, pyrrole,thiophenes, furans, imidazoles, and the like, as well as fused ringstructures having rings of different sizes, such as benzofurans,indoles, purines, and the like.

Also included within the scope of the present invention are alicyclicgroups, as that term is understood in the art, and heterocyclic groups.As used herein, the term “heterocyclic group” refers to non-aromaticcyclic substituents in which one or more members of the ring is notcarbon, for example oxygen, sulfur or nitrogen.

The terms “alkylaryl” (or “alkaryl”) or “alkylheteroaryl” as used hereinrefer to groups having an alkyl moiety attached to an aryl or heteroarylring. The alkyl moiety is preferably a straight, branched or cyclicalkyl group having one to about six carbon atoms. This alkyl moiety mayalso contain oxygen, nitrogen or sulfur, and therefore may be an alkoxygroup. The aryl or heteroaryl moiety of the alkylaryl group is asubstituted or unsubstituted aryl or heteroaryl group, as these termsare described above. As used herein, the terms “alkylaryl” or“alkylheteroaryl” will also be used to refer to arylalkyl groups orheteroarylalkyl groups, as those terms are understood in the art, anddenotes attachment of such a substituent at either the alkyl or the arylportion of the group. Thus, for example, a benzyl group would beembraced by the term “alkylaryl”.

Any of the cyclic substituents described above, such as the aryl,heteroaryl, alkylaryl, alkylheteroaryl, alicyclic, or heterocyclicgroups are optionally substituted with one or more substituents aslisted above. In the case of more than one substituent, the substituentsare independently selected. “Alkoxy groups” and “alkyl groups” includestraight or branched chains having up to about ten members. “Halogen”refers to chlorine, bromine, iodine and fluorine. “Aryl and heteroarylgroups” are as described above. When a carboxylic acid is a substituent,it will be appreciated that the moiety represents an acid such asbenzoic acid. As used herein, the termheterocycloaryl-carbonyl-L-glutamate group may include for example athiophene-carbonyl-L-glutamate group, a furan-carbonyl-L-glutamategroup, a pyrrole-carbonyl-L-glutamate group, and apyridine-carbonyl-L-glutamate group, and the termheterocycloalkyl-carbonyl-L-glutamate group may include for example adihydrothiophene-carbonyl-L-glutamate group, atetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof, asthose terms are understood by one skilled in the art.

As used herein, the terms “aroyl” or “heteroaroyl”, such as when usedwithin the term p-aroyl-L-glutamate, refers to benzoyl, napthoyl,thiophenoyl, furophenoyl, pyrroyl, and any other “aroyl” or“heteroaroyl” as these terms are understood by one skilled in the art.“Aroyl” and“heteroaroyl” are generally defined in the art as an aromaticor heteroaromatic compound having a carbonyl moiety. As used herein, theterm “glutamate” will be understood as representing both the ester form(glutamate) and the acid form (glutamic acid).

Those skilled in the art shall understand that chemical structure ofFormula II is a preferred example of this invention and that Formula IIis a tautomer of an embodiment of a compound of Formula I. Those skilledin the art understand that chemical structures are often drawn as onetautomeric form over another. This invention provides for severaltautomeric forms as covered by the description of Formula I. Thetautomeric forms taught by Formula I provide several structuralembodiments that will be appreciated by those skilled in the art, suchas for example the compounds having Formula II.

Proliferative diseases and/or disorders that may be treated according tothe methods of the present invention include, without limitation,ovarian cancer, endometrial and cervical cancer, renal cancer, andbreast cancer, and automimune diseases such as for example rheumatoidarthritis.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a compound ofthe present invention to produce the desired effect in association witha pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the particularcompound and the particular effect, or therapeutic response, that isdesired to be achieved.

Compounds of Formula I or II, or pharmaceutically acceptable salts,prodrugs, solvates, or hydrates thereof, can be administered to apatient (an animal or human) via various routes including parenterally,orally or intraperitoneally. Parenteral administration includes thefollowing routes that are outside the alimentary canal (digestivetract): intravenous; intramuscular; interstitial, intraarterial;subcutaneous; intraocular; intracranial; intraventricular;intrasynovial; transepithelial, including transdermal, pulmonary viainhalation, ophthalmic, sublingual and buccal; topical, includingdermal, ocular, rectal, or nasal inhalation via insufflation ornebulization. Specific modes of administration shall depend on theindication. The selection of the specific route of administration andthe dose regimen is to be adjusted or titrated by the clinicianaccording to methods known to the clinician in order to obtain theoptimal clinical response. The amount of compound to be administered isthat amount which is therapeutically effective. The dosage to beadministered to a patient shall depend on the characteristics of thepatient being treated, including for example, but not limited to, thepatient's age, weight, health, and types and frequency of concurrenttreatment, if any, of any other chemotherapeutic agent(s), all of whichis determined by the clinician as one skilled in the art.

Compounds of Formula I or II, or a pharmaceutically acceptable salt,prodrug, solvate or hydrate thereof, that are orally administered can beenclosed in hard or soft shell gelatin capsules, or compressed intotablets. Compounds also can be incorporated with an excipient and usedin the form of ingestible tablets, buccal tablets, troches, capsules,sachets, lozenges, elixirs, suspensions, syrups, wafers and the like.Compounds of Formula I or II can be in the form of a powder or granule,a solution or suspension in an aqueous liquid or non-aqueous liquid, orin an oil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch; gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoring.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic. Additionally, thecompounds of Formulas I, II, or a pharmaceutically acceptable salt,prodrug, solvate or hydrate of Formulas I or II, can be incorporatedinto sustained-release preparations and formulations.

The compounds of Formula I, II, or a pharmaceutically acceptable salt,prodrug, solvate or hydrate thereof, can be administered to the centralnervous system, parenterally or intraperitoneally. Solutions of thecompound as a free base or a pharmaceutically acceptable salt can beprepared in water mixed with a suitable surfactant, such ashydroxypropylcellulose. Dispersions also can be prepared in glycerol,liquid polyethylene glycols and mixtures thereof, and in oils. Underordinary conditions of storage and use, these preparations can contain apreservative and/or antioxidants to prevent the growth of microorganismsor chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compounds of the present invention may be contained within, mixed with,or associated with, a suitable (acceptable) pharmaceutical carrier foradministration to a patient according to the particular route ofadministration desired. Suitable or acceptable pharmaceutical carriersrefer to any pharmaceutical carrier that will solubilize the compoundsof the present invention and that will not give rise to incompatabilityproblems, and includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such suitable or acceptablepharmaceutical carriers are well known by those skilled in the art.Preferred carriers include sterile water, physiologic saline, and fivepercent dextrose in water. Examples of other suitable or acceptablepharmaceutical carriers include, but are not limited to, ethanol, polyol(such as propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, or vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size (in the case of adispersion) and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating a compound ofFormula I or II in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the sterilized compound of Formula I or II into a sterilevehicle that contains the basic dispersion medium and any of the otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of Formula I or II, as described herein, canbe administered to a patient alone or in combination withpharmaceutically acceptable carriers or as pharmaceutically acceptablesalts, solvates or hydrates thereof, the proportion of which isdetermined by the solubility and chemical nature of the compound, chosenroute of administration to the patient and standard pharmaceuticalpractice.

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLES

FIG. 2 shows the biological effects of various compounds of the presentinvention, namely, Samples: AAG 154353, AAG154360, AAG154484, AAG154468,AAG154479, and AAG154489. These compounds were evaluated forcytotoxicity towards assorted cell lines, namely, KB human tumor cellsexpressing FRs and RFC, PC43-10, and Chinese hamster ovary expressingRFC, and RT16 Chinese hamster ovary cells expressing FRs but no RFC.FIG. 2 shows the IC₅₀ of each of the Sample compounds of the presentinvention towards each cancer cell line. The IC₅₀ is the inhibitoryconcentration required to effectuate fifty percent inhibition of cellgrowth.

Compounds AAG154353, AAG154360, AAG154489 and AAG154468 are potentinhibitors of KB human tumor cells known to express high levels offolate receptor alpha (FRα) with values of 0.25, 3.4, 0.3 and 122 nMrespectively (see FIG. 2). In addition, the compounds are potentlyinhibitory against RT16 cells that express FRα and D4 cells that expressfolate receptor beta (FRβ). All the compounds as anticipated wereinactive against cells engineered to lack FRα or Fβ, such as PC43 (onlyRFC) and R₂ cells that lack FRα or β, as well as RFC. These results showthat compounds AAG154353, AAG154360, AAG145489 and AAG154468 areselectively inhibitory only against cells and tumor cells that expressFRs.

Inhibitory effects of AAG154353 and AAG154360, as examples of theheteroaroyl side chain analogs of the compounds of the present inventionof Formula I, on RFC transport and FR binding were also evaluated. ThePC43 cells, (RFC containing cells) that express RFC only, were used andthe ability of the compounds to inhibit [³H] MTX (methotrexate) uptake.Both example compounds AAG154353 and AAG154360 had very poor inhibitoryactivity, <20%, showing that these compounds do not inhibit [³H] MTXuptake in PC43 cells. In contrast, the evaluation of the FRα bindingaffinity compared to folic acid showed very high binding affinities withrelative affinities similar to folic acid set to a value of 1 (see FIG.2). Similar results were obtained with FRβ and show that the prototypeanalogs are selective for and are excellent substrates for FRα and FRβ.

In-Vivo Evaluation of Sample: AAG154353 in Advanced Tumor Xenograft

SCID female mice bearing advanced stage human KB cervical tumors asxenografts (xenograft tumors) were administered compound AAG154353 ofthe present invention intravenously at various doses and schedules.

Mice were maintained on a Folic Acid deficient diet (Harlan-Teklad Diet#00434; Madison, Wis.) exclusively for approximately three weeks at thestart of treatment. Three mice bearing late stage KB tumor xenografts(446 to 650 mg at the start of treatment) were each treated on variousdose/schedules with AAG154353 IV. In all three cases, tumors regressedcompletely. Thus, significant antitumor activity was detected. Toxicityas reflected in weight loss was modest.

Dose/schedules tested:

-   1) Q3d×4 does starting day 37; 750 mg/kg total dose—mouse tumor free    for one month, 30 days;-   2) Q2d×4 doses starting day 43; 500 mg/kg total dose—mouse with 63    mg tumor; tumor free up to day 60.-   3) Q4d×3 doses starting day 48; 187.5 mg/kg total dose—mouse tumor    free for one month, 30 days.    Description of FR and Human Proton-Coupled Folate Transporter    (hPCFT) Studies with Pyrrolo[2,3-d]pyrimidine Antifolates-   Folate receptor studies: Following the inventors' initial studies of    pyrrolo[2,3-d]pyrimidine antifolates with 1 or 3 to 6 carbon bridge    substitutions as represented by Compound AAG120366-2, structure    shown in FIG. 3) to identify FR targeted agents with low level    transport by human reduced folate carrier (hRFC) (Deng et al.,    2008), we tested compounds of Formula I of the present invention,    namely Example compounds AAG154353 and AAG154360, each having a    thienoyl side chain in KB human tumor cells (see Table 1). The    compounds AAG154353 and AAG154360 were initially tested for their    growth inhibitory effects against KB human tumor cells, which    express FR alpha and hRFC but insignificant levels human proton    coupled folate transporter (hPCFT), using a fluorescence-based    (“Cell Titer-blue”) cytotoxicity screen. In KB cells, IC50s of 0.25    and 3.4 nM were measured from compounds AAG154353 and AAG154360.    FR-targeted activity by AAG154353 and AAG154360 was confirmed by    co-treatments with folic acid (200 nM) which completely reversed    growth inhibition of these agents. AAG154353 and AAG154360 were also    tested in isogenic Chinese hamster ovary (CHO) sublines, engineered    to express human FR-(RT16) or hRFC (PC43-10). For PC43-10, results    were compared to those for hRFC- and FR-null R₂ CHO cells from which    they were derived, whereas those for RT16 cells were compared to    those for a parallel incubation in the presence of an elevated    concentration of folic acid, as with the KB cells. AAG154353 and    AAG154360 showed a high level of FR-targeted activity toward RT16    cells. Neither AAG154353 and AAG154360 showed appreciable growth    inhibition of hRFC-expressing PC43-10 cells. These results are    summarized in Table 1.

TABLE 1 IC50s (nM) for antifolate analogs in cell proliferationinhibition of RFC- and FR- expressing cell lines and results of in vitroand in situ GARFTase assays. Experiments were performed in standardRPMI1640/10% dialyzed fetal bovine serum. Growth inhibition results arepresented as mean values IC50 values (nM) from 2-3 experiments. ForGARFTase assays, results are shown as mean IC50s. SEM values are shownin parentheses. Growth inhibition (IC50, nM) hFRα hRFC/FRα GARFTaseAssay hRFC RT16 KB In vitro IC50 In situ Antifolate PC43-10 R2 RT16(+FA) KB (+FA) (μM) IC50 (nM) AAG1203662 304 448 4.1 >1000 1.7 >10002.44 (0.12) 18 (2) AAG154353 >1000 >1000 2 >1000 0.25 >1000 0.06 (0.004)0.63 (0.52) AAG154360 >1000 >1000 2 >1000 3.4 >1000 3.31 (0.32) 7.65(3.7) Methotrexate 12 216 114 461 6.0 20 — Pemetrexed 138 894 42 388 68327 >20 30 (7.7) Raltitexed 6.3 >1000 15 >1000 5.9 22 — Lometrexol12 >1000 12 188 1.2 31 0.78 (0.08) 14 (5.6) Trimetrexate 25 6.7 13 4.158 155 — GW1843U89 11 >1000 277 >1000 5.8 32 —

-   Human proton-coupled folate transporter (hPCFT) studies. Following    reports of a novel pH transporter termed PCFT in the proximal small    intestine and possibly solid tumors, the inventors' established an    expression profile of this transporter compared to hRFC and FRα in a    wide range of cell lines derived from human solid tumors and    leukemias. mRNA levels for hPCFT, hRFC and FRα were measured by    real-time RT-PCR (qPCR) and normalized to levels of    glyceraldyde-3-phosphate dehydrogenase. Results shown in FIG. 4    clearly demonstrate appreciable hPCFT transcripts in a large number    of human solid tumor cell lines of different origins (e.g., breast,    prostate, ovarian, etc.) and uniformly low level hPCFT in human    leukemias. hPCFT levels were highest in SKOV3 (ovarian), HepG2    (hepatoma), HeLa (cervical), and T47D (breast) cancer cells. hRFC    transcripts were detected in all cell lines with exception of HeLa    R₅ and MDA-MB-231 (both documented to express low to undetectable    hRFC). FRα was only detected in a small subset of ovarian, cervical,    and breast cell lines (inset). FIG. 4 sets forth hRFC and hPCFT    transcript levels in solid tumor and leukemia cell lines.    Transcripts levels were measured by qPCR from total RNAs using a    Roche 480 Lightcycler and Sybr Green1 detection. hRFC/hPCFT    transcript levels were normalized to GAPDH transcripts. The inset    (black bars) shows results for solid tumors in which FR alpha    transcripts could be detected. FR alpha could not be detected in the    remaining solid tumors and all of the leukemia cells. The tumor    types for the solid tumors shown in FIG. 4 are as follows: DU-145,    PC-3 (prostate); UCVA-1 (pancreas); SKOV3, Ovcar-3, Igrov-1 (ovary);    HTB139 (muscle); H2591, MET5A, H2452, H2313 (mesothelioma);    SK-MEL-28 (melanoma); HepG2 (hepatoma); HT1080 (fibrosarcoma); Y79    (eye); SW-620, HCG-116 (colon); HeLa, R5, KB (cervical); T-47D,    MDA-MB435, MDA-MB231, MCF-7 (breast); SK-N-MC (brain); and HTB166,    TE-85 (bone).

To study the functional and biochemical properties of hPCFT, theinventors prepared a myc-his6 tagged hPCFT (hPCDT ^(myc-his6))cDNAconstruct by RT-PCR from RNA prepared from wild type (wt) HeLa cells.hPCFT^(myc-his6) (in pCDNA3.1 plasmid) was transiently expressed in R₅HeLa cells (expresses some low level of hPCFT), and assayed forhPCFT^(Myc-his6) protein on westerns with Myc-specific antibody andtransport activity at pH 5.5 for comparison with mock (vector control)transfected R5 cells (FIG. 5, upper panel). To generate stabletransfectants, PCFT- and RFC-null R2 CHO cells were electroporated withthe hPCFT^(myc-his6)-pCDNA3.1 construct. Cells were selected with G418and screened for hPCFT^(myc-his6) protein. The best clone (designatedR₂-hPCFT#4) was further characterized (FIG. 5, lower panel). A highlevel of hPCFT^(Myc-His6) protein was detected on Western blots(westerns) accompanying significant [³H]Mtx transport at pH 5.5.Transport was negligible at pH 7.2-7.4 (not shown). For hRFC-expressingpC43-10 cells, [³H]MTX transport was active at pH 7.2-7.4 but wasundetectable at pH 5.5 (data not shown). FIG. 5 shows transfection ofHeLa R₅ and CHO R₂ Cells with hPCFTMyc-his10. Cells were transfectedwith hPCFTMyc-his6 either transiently (HeLa) or stably (R₂).PCFTMyc-His10 protein was measured on westerns with Myc specificantibody (insets) and hPCFT activity was measured in MES-buffered salineat pH 5.5 with 1 micromolar 3H-Mtx as substrate.

In growth inhibition assays, the inventors found that sensitivities toassorted classical antifolates including methotrexate (MTX), GW1843U89,lometrexol, pemetrexed, PT523, and raltitrexed forhPCFT^(Myc-his6)-expressing R2-hPCFT #4 cells and hRFC-expressing (˜4 to8-fold and ˜10- to 150-fold, respectively), in comparison with vectorcontrol R2 cells (Tables 1 and 2). Thus, even at the relatively neutralpH (˜7.2) of tissue culture media, classical antifolates appear to besubstrates for hPCFT, as reflected in patterns of growth inhibition inthe hPCFT-transfected and mock transfected CHO cells. By comparingresults with R₂-hPCFT#4 and pC43-10 cells, only the antifolate PT523 wascompletely selective in its effects toward hRFC over hPCFT, whereas noneof these agents showed significant selectivity toward hPCFT over hRFC.

The compounds of Formula I of the present invention, for example, havestructures with modifications in several regions, including but notlimited to the pteridine moeity, the carbon bridge region of the sidechain, the p-aminobenzoic acid, or terminal glutamate, and combinationsthereof. For example, by standard growth inhibition assays, weidentified a novel 6-substituted pyrrolo[2,3 -d]pyrimidine antifolate,namely AAG154353, with striking sensitivity toward R₂-hPCFT#4 cells(-20- to 30-fold greater than those for vector-control R₂ cells) (Table2) and a nearly complete lack of drug activity toward hRFC-expressingpC43-10 cells (Table 1). Specificity for hPCFT over hRFC was furthersuggested by assays of direct competition with [³H]MTX for cellularuptake by R₂-hPCFT#4 (FIG. 6). In both transport and cytotoxicityexperiments, compound AAG154353 exhibited potencies similar to that forpemetrexed, the best substrate yet described for hPCFT. However, thehRFC substrate, PT523, was completely inert in inhibiting cellproliferation or [³H]MTX uptake with R₂-hPCFT#4 cells. In contrast topemetrexed, compound AAG154353 showed negligible inhibition of [³H]MTXtransport from hRFC in pC43-10 cells (at pH 7.2-7.4) (not shown).

Identification of intracellular enzyme target Compound AAG120366-2 waspreviously reported by this applicant to potently inhibit GARFTase, thefirst folate-dependent step in the de novo purine biosynthetic pathway(Deng et al., 2008). To localize the probable enzyme target(s) forcompound AAG154353, we tested the growth inhibitory effects of thiscompounds toward KB in the presence of adenosine (60 μM) or thymidine(10 μM). Thymidine (10 μM) did not alter the growth inhibitory effectsof AAG154353 whereas adenosine (60 μM) was completely protective, thusestablishing the de novo purine biosynthetic pathway as the primarytarget. Compound AAG154353 was completely protected by5-amino-4-imidazolecarboxamide (AICA) (320 μM) which identified GARFTaseas the likely intracellular target. We used in vitro and in situGARFTase inhibition assays with antifolate analogs to confirminhibitions of this enzyme target. With the purified recombinant mouseGARFTase, compounds AAG154353 and AAG154360 were inhibitory with IC50sof 0.06 μM and 3.31 μM, respectively (Table 1). In the in situ GARFTaseassay, incorporation of ¹⁴C-glycine into formyl glycinamideribonucleotide was measured and was inhibited at nM concentrations ofthe drugs, with the most potent effects by inhibitor AAG154353 (Table1). The dramatic differences in inhibition potencies by in situ versusin vitro assays of GARFTase for all these agents likely reflect anexacerbation of enzyme binding affinities by drug polyglutamates withincells.

TABLE 2 Growth inhibition by antifolate drugs toward hPCFT- and hRFCstable R2 CHO transfectants. Analogs R2-hPCFT#4 R2/VC Methotrexate143.25 ± 29.4  >1000 GW1843U89 39.23 ± 7.31  135.76 ± 13.99  Lometrexol183.28 ± 12.61  >1000 Pemetrexed 27.51 ± 3.26  150.42 ± 22.54 PT523 >1000 >1000 Raltitrexed 63.76 ± 3.26  604.39 ± 53.63  AAG120366-229.14 ± 4.72  490.89 ± 77.58  AAG154353 48.25 ± 17.75 >1000 Experimentswere performed in folate-free RPMI1640/10% dialyzed fetal bovine serumsupplemented with 25 nM leucovorin. Growth inhibition was measured by aflorescence (Cell TiterBlue)-based assay after 96 h of exposure to arange of inhibitor concentrations. Results are presented as 50%inhibitory concentrations. (IC50's)

Our results establish a high frequency of expression of hPCFT in solidtumors over leukemias. hRFT is expressed in both solid tumors andleukemias whereas FRα is expressed exclusively in a subset of solidtumors. Our results with compound AAG154353 are unprecedented in thatthey are the first to exhibit a unique and selective binding to FRα andhPCFT over hRFC that results in a potent growth inhibition even atphysiologic pH. Given the acidic pH optimum for hPCFT (pH 5.5-6.8),these growth inhibitory effects are clearly exascerbated at lower pHvalues (6.5-6.8) as occurs, for example, in a solid tumor environment.Indeed, our initial studies over a range of pH values establish a2-3-fold increased inhibition of hPCFT transport at acid (ph<7) overneutral (pH>7) conditions.

The development of novel small molecule cytotoxins such as the compoundsof Formula I of the present invention that are selectively transportedby hPCFT provide exciting new therapeutic applications for solid tumortargeting. This is based on the notion of effectively “highjacking” anessential biological characteristic of solid tumors, namely their acidicmicroenvironment, for selective delivery of the cytotoxic compounds ofthe present invention.

Synthesis of Compounds Chemistry

Target compounds AAG154353, AAG154360 and AAG154484 were synthesized asshown in Scheme 1. Commercially available pent-4-ynoic acid 1a orhex-5-ynoic acid 1b or hept-6-ynoic acid 1c (Scheme 1) was converted tothe acid chlorides 2a-c and immediately reacted with diazomethanefollowed by 48% HBr to give the desired α-bromomethylketones 4a-c.Condensation of 2,4-diamino-6-hydroxypyrimidine with 4a-c at roomtemperature for 3 days afforded the 6-substitutedpyrrolo[2,3-d]pyrimidines 5a-c (51-74% yield). Compounds 6a-c wereobtained by a Sonogashira coupling of 5a-c with(S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethylester 9. Hydrogenation and saponification of 6a-c afforded AAG154353,AAG154360 and AAG154484, respectively. Compound 9 (Scheme 1) wassynthesized by coupling the commercially available5-bromo-2-thiophene-carboxylic acid 8 and L-glutamate diethyl esterhydrochloride in 72% yield.

-   Intermediates for compounds AAG154468 and AAG154479 were synthesized    as shown in Scheme 2. Direct saponification of 6a and 6c afforded    AAG154468 and AAG154479 in 95% yield.

-   AAG154489 was synthesized as shown in Scheme 3. Compounds 10 was    obtained by a Sonogashira coupling of 5a (from scheme 1) with    (S)-2-[(5-bromo-furan-2-carbonyl)-amino]-pentanedioic acid diethyl    ester 13. Compound 13 (Scheme 3) was synthesized by coupling the    commercially available 5-bromo-2-furan-carboxylic acid 12 and    L-glutamate diethyl ester hydrochloride in 72% yield. Hydrogenation    and sanonification of 10 afforded AAG154489.

-   AAG154485 was synthesized as shown in Scheme 4. Direct    saponification of 10 afforded AAG154485 in 95% yield.

Experimental Section

-   All evaporations were carried out in vacuo with a rotary evaporator.    Analytical samples were dried in a CHEM-DRY vacuum (0.2 mm Hg)    drying oven over P₂O₅. Melting points were determined on a MELTEMP    II melting point apparatus with FLUKE 51 K/J electronic thermometer    and are uncorrected. NMR spectra for proton (¹H) were recorded on a    Bruker WH-300 (300 MHz) spectrometer. The chemical shift values are    expressed in ppm (parts per million) relative to tetramethylsilane    as internal standard; s=singlet, d=doublet, t=triplet, q=quartet,    m=multiplet, br=broad singlet. The relative integrals of peak areas    agreed with those expected for the assigned structures. Thin-layer    chromatography (TLC) was performed on PE SIL G/UV silica gel plates    with fluorescent indicator, and the spots were visualized under 254    and 365 nm illumination. Proportions of solvents used for TLC are by    volume. Column chromatography was performed on 230-400 mesh silica    gel purchased from Fisher, Somerville, N.J. Elemental analyses were    performed by Atlantic Microlab, Inc., Norcross, Ga. Element    compositions are within ±0.4% of the calculated values. Fractional    moles of water or organic solvents frequently found in some    analytical samples of antifolates were not prevented despite 24-48 h    of drying in vacuo and were confirmed where possible by their    presence in the ¹H NMR spectra. High resolution mass spectrometry    (HRMS) was performed on a Waters Q-TOF (API-US) by Department of    Chemistry, University of Pittsburgh, Pittsburgh, Pa. All solvents    and chemicals were purchased from Aldrich Chemical Co. and Fisher    Scientific and were used as received.

General Procedure for the Synthesis of Compounds 5a-c

To 1a-c (10 mmol) in a 250 mL flask were added oxalyl chloride (7.61 g,60 mmol) and anhydrous CH₂C₂ (20 mL). The resulting solution wasrefluxed for 1 h and then cooled to room temperature. After evaporatingthe solvent under reduced pressure, the residue 2a-c were dissolved in20 mL of Et₂O. The resulting solution was added drop wise to anice-cooled diazomethane (generated in situ from 15 g diazald by usingAldrich Mini Diazald Apparatus) in an ice bath over 10 min. Theresulting mixture was allowed to stand for 30 min and then stirred foran additional 1 h. To this solution was added 48% HBr (20 mL). Theresulting mixture was refluxed for 1.5 h. After cooling to roomtemperature, the organic layer was separated and the aqueous layerextracted with Et₂O (200 mL×2). The combined organic layer and Et₂Oextract was washed with two portions of 10% Na₂CO₃ solution and driedover Na₂SO₄. Evaporation of the solvent afforded 4a-c in 94% yield. To asuspension of 2,6-diaminopyrimidin-4-one (1.26 g, 10 mmol) in anhydrousDMF (25 mL) was added 4a-c (about 9.4 mmol). The resulting mixture wasstirred under N₂ at room temperature for 3 days. After evaporation ofsolvent under reduced pressure, MeOH (20 mL) was added followed bysilica gel (5 g). The resulting plug was loaded on to a silica gelcolumn (3.5×12 cm) and eluted with CHCl₃ followed by 3% MeOH in CHCl₃and then 5% MeOH in CHCl₃. Fractions with an R_(f)=0.58 (TLC) werepooled and evaporated to afford 5a-c as white powder.

2-amino-6-but-3-ynyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-4-one (5a)

Compound 5a was prepared using the general method described for thepreparation of 5a-c, from pent-4-ynoic acid 1a (0.98 g, 10 mmol) to give1.4 g (74%) of 5a as white powder. mp 230-231° C.; ¹H NMR (DMSO-d₆): δ2.41-2.45 (m, 2H), 2.64-2.67 (m, 2H), 2.77 (t, J=2 Hz, 1H), 5.93 (s,1H), 5.98 (s, 2H), 10.13 (s, 1H), 10.81 (s, 1H). HRMS calcd forC₁₀H₁₀N₄O (M⁺), 203.0933; found: 203.0925.

2-amino-6-pent-4-ynyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-4-one (5b)

Compound 5b was prepared using the general method described for thepreparation of 5a-c, from hex-5-ynoic acid 1b (1.12 g, 10 mmol) to give1.1 g (51%) of 5b as white powder. mp 233-234° C.; ¹H NMR (DMSO-d₆): δ1.64-1.79 (m, 2H), 2.14-2.20 (m, 2H), 2.58-2.61 (t, J=10 Hz, 2H),2.80-2.82 (t, J=3.2 Hz, 1H), 5.95 (s, 1H), 6.53 (s, 2H), 10.70 (s, 1H),11.14 (s, 1H).

2-amino-6-hex-5-ynyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-4-one (5c)

Compound 5c was prepared using the general method described for thepreparation of 5a-c, from hept-6-ynoic acid 1c (1.26 g, 10 mmol) to give1.43 g (62%) of 5c as white powder. mp 236-237° C.; ¹H NMR (DMSO-d₆): δ1.40-1.47 (m, 2H), 1.52-1.67 (m, 2H), 2.13-2.17 (m, 2H), 2.46 (m, 2H),2.75-2.77 (m, 1H), 5.87 (s, 1H), 6.16 (s, 2H), 10.31 (s, 1H), 10.90 (s,1H).

General Procedure for the Synthesis of Compounds 6a-c

To a 250-mL round-bottomed flask, equipped with a magnetic stirrer andgas inlet, were added a mixture of tetrakis(triphenylphosphine)palladium(0) (185 mg, 0.16 mmol), triethylamine(1.01 g, 10 mmol),(S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethylester 9 (588 mg, 1.5 mmol) and anhydrous DMF (20 mL). To the stirredmixture, under N₂, was added copper(I) iodide (30 mg, 0.16 mmol) and5a-c (1 mmol), and the reaction mixture was stirred at room temperatureovernight (17-18 h). After evaporation of solvent under reducedpressure, MeOH (20 mL) was added followed by silica gel (5 g). Theresulting plug was loaded on to a silica gel column (3.5×12 cm) andeluted with CHCl₃ followed by 3% MeOH in CHCl₃ and then 5% MeOH inCHCl₃. Fractions with an R_(f)=0.53 (TLC) were pooled and evaporated toafford 6a-c as brown powder.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-but-1-ynyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (6a)

Compound 6a was prepared using the general method described for thepreparation of 6a-c, from 5a (202 mg, 1 mmol) to give 386 mg (75%) of 6aas brown powder. mp 81-82° C.; ¹H NMR (DMSO-d₆): δ 1.16-1.21 (m, 6H),1.93-2.15 (m, 2H), 2.40-2.45 (t, J=10 Hz, 2 H), 3.06-3.15 (m, 4H),4.01-4.15 (m, 4H), 4.35-4.43 (m, 1H), 6.00 (s, 1H), 6.04 (s, 2H),7.22-7.23 (d, J=5.2 Hz, 1H), 7.77-7.78 (d, J=5.2 Hz, 1H), 8.83-8.85 (d,J=10 Hz, 1H), 10.18 (s, 1H), 10.89 (s, 1H). HRMS calcd for C₂₄H₂₇N₅O₆S(M⁺), 514.1760; found: 514.1753.

(S)-2-({5-[5-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-pent-1-ynyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (6b)

Compound 6b was prepared using the general method described for thepreparation of 6a-c, from 5b (216 mg, 1 mmol) to give 380 mg (72%) of 6bas brown powder. mp 84-85° C.; ¹H NMR (DMSO-d₆): δ 1.16-1.20 (m, 6H),1.81-1.90 (m, 2H), 1.92-2.13 (m, 2H), 2.40-2.45 (t, J=10 Hz, 2H), 2.46(m, 2H), 2.59-2.64 (t, J=9.6 Hz, 2H), 4.00-4.14 (m, 4H), 4.35-4.43 (m,1H), 5.91 (s, 1H), 5.99 (s, 2H), 7.24-7.25 (d, J=5.2 Hz, 1H), 7.77-7.78(d, J=5.2 Hz, 1H), 8.82-8.84 (d, J=10 Hz, 1H), 10.14 (s, 1H), 10.86 (s,1H).

(S)-2-({5-[6-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-hex-1-ynyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (6c)

Compound 6c was prepared using the general method described for thepreparation of 6a-c, from 5c (230 mg, 1 mmol) to give 396 mg (73%) of 6cas brown powder. mp 85-86° C.; ¹H NMR (DMSO-d₆): δ 1.13-1.19 (m, 6H),1.48-1.57 (m, 2H), 1.62-1.71 (m, 2H), 1.91-2.12 (m, 2H), 2.39-2.43 (t,J=7.6 Hz, 2H), 2.46-2.48 (m, 4H), 4.00-4.12 (m, 4H), 4.34-4.40 (m, 1H),5.87 (s, 1H), 5.96 (s, 2H), 7.23-7.24 (d, J=3.6 Hz, 1H), 7.75-7.76 (d,J=3.6 Hz, 1H), 8.80-8.82 (d, J=7.6 Hz, 1H), 10.12 (s, 1H), 10.81 (s,1H).

General Procedure for the Synthesis of Compounds 7a-c

To a Parr flask were added 6a-c (0.75 mmol), 10% palladium on activatedcarbon (120 mg), and MeOH (100 mL). Hydrogenation was carried out at 55psi of H₂ for 4 h. The reaction mixture was filtered through Celite,washed with MeOH (100 mL) and concentrated under reduced pressure togive 7a-c as yellow powder.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-butyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (7a)

Compound 7a was prepared using the general method described for thepreparation of 7a-c, from 6a (386 mg, 0.75 mmol) to give 369 mg (95%) of7a as yellow powder. mp 74-75° C.; ¹H NMR (DMSO-d₆): δ 1.13-1.20 (m,6H), 1.62 (m, 4H), 1.89-2.13 (m, 2H), 2.39-2.44 (t, J=10 Hz, 2H), 2.69(m, 2H), 2.81 (m, 2H), 4.00-4.13 (m, 4H), 4.34-4.42 (m, 1H), 5.85 (s,1H), 5.97 (s, 2H), 6.88-6.89 (d, J=4.8 Hz, 1H), 7.67-7.68 (d, J=4.8 Hz,1H), 8.60-8.63 (d, J=10 Hz, 1H), 10.13 (s, 1H), 10.81 (s, 1H). HRMScalcd for C₂₄H₃₁N₅O₆S (M⁺), 518.2073; found: 518.2077.

(S)-2-({5-[5-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-pentyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (7b)

Compound 7b was prepared using the general method described for thepreparation of 7a-c, from 6b (380 mg, 0.72 mmol) to give 360 mg (94%) of7b as yellow powder. mp 77-78° C.; ¹H NMR (DMSO-d₆): δ 1.12-1.21 (m,8H), 1.53-1.67 (m, 4H), 1.91-2.14 (m, 2H), 2.39-2.43 (t, J=10 Hz, 2H),2.46 (m, 2H), 2.77-2.82 (t, J=9.6 Hz, 2H), 4.00-4.14 (m, 4H), 4.33-4.41(m, 1H), 5.84 (s, 1H), 5.97 (s, 2H), 6.88-6.89 (d, J=4.8 Hz, 1H),7.68-7.69 (d, J=4.8 Hz, 1H), 8.60-8.63 (d, J=10 Hz, 1H), 10.12 (s, 1H),10.79 (s, 1H).

(S)-2-({5-[6-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-hexyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (7c)

Compound 7c was prepared using the general method described for thepreparation of 7a-c, from 6c (396 mg, 0.73 mmol) to give 379 mg (94%) of7c as yellow powder. mp 78-79° C.; ¹H NMR (DMSO-d₆): δ 1.15-1.19 (m,6H), 1.23-1.31 (m, 4H), 1.47-1.68 (m, 4H), 1.90-2.14 (m, 2H), 2.37-2.48(m, 6H), 4.02-4.12 (m, 4H), 4.33-4.41 (m, 1H), 5.84 (s, 1H), 5.96 (s,2H), 7.08-7.09 (d, J=3.6 Hz, 1H), 7.78-7.79 (d, J=3.6 Hz, 1H), 8.72-8.74(d, J=7.6 Hz, 1H), 10.11 (s, 1H), 10.79 (s, 1H).

General Procedure for the Synthesis of Target Compounds AAG154353,AAG154360, AAG154484, AAG154468, AAG154479, AAG154489 and AAG154485

To a solution of 7a-c (0.7 mmol) in MeOH (10 mL) was added 1 N NaOH (10mL) and the mixture was stirred under N₂ at room temperature for 16 h.TLC showed the disappearance of the starting material (R_(f)=0.45) andone major spot at the origin (MeOH/CHCl₃ 1:5). The reaction mixture wasevaporated to dryness under reduced pressure. The residue was dissolvedin water (10 mL), the resulting solution was cooled in an ice bath, andthe pH was adjusted to 3-4 with drop wise addition of 1 N HCl. Theresulting suspension was frozen in a dry ice-acetone bath, thawed to4-5° C. in the refrigerator, and filtered. The residue was washed with asmall amount of cold water and dried in vacuum using P₂O₅ to afford thetarget compounds as white powder.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-butyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154353)

Compound AAG154353 was prepared using the general method described forthe preparation of target compounds, from 7a (369 mg, 0.71 mmol) to give312 mg (95%) of AAG154353 as white powder. mp 179-180° C.; ¹H NMR(DMSO-d₆): δ 1.62 (m, 4H), 1.91-2.05 (m, 2H), 2.31-2.36 (t, J=7.4 Hz,2H), 2.69 (m, 2H), 2.81 (m, 2H), 4.29-4.43 (m, 1H), 5.87 (s, 1H), 6.10(s, 2H), 6.87-6.88 (d, J=4 Hz, 1H), 7.67-7.68 (d, J=4 Hz, 1H), 8.49-8.52(d, J=8 Hz, 1H), 10.26 (s, 1H), 10.88 (s, 1H), 12.42 (br, 2H). HRMScalcd for C₂₀H₂₃N₅O₆S (M⁺), 462.1447; found: 462.1462.

(S)-2-({5-[5-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-pentyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154360)

Compound AAG154360 was prepared using the general method described forthe preparation of target compounds, from 7b (360 mg, 0.68 mmol) to give306 mg (95%) of AAG154360 as white powder. mp 181-182° C.; ¹H NMR(DMSO-d₆): δ 1.28-1.34 (m, 2H), 1.54-1.66 (m, 4H), 1.85-2.10 (m, 2H),2.30-2.34 (t, J=7.4 Hz, 2H), 2.43-2.47 (t, J=7.6 Hz, 2H), 2.76-2.80 (t,J=7.6 Hz, 2H), 4.29-4.43 (m, 1H), 5.83 (s, 1H), 5.95 (s, 2H), 6.86-6.87(d, J=4 Hz, 1H), 7.66-7.67 (d, J=4 Hz, 1H), 8.49-8.51 (d, J=8 Hz, 1H),10.11 (s, 1H), 10.78 (s, 1H), 12.32 (br, 2H). HRMS calcd for C₂₁H₂₅N₅O₆S(M⁺), 476.1604; found: 476.1617.

(S)-2-({5-[6-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-hexyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154484)

Compound AAG154484 was prepared using the general method described forthe preparation of target compounds, from 7c (379 mg, 0.69 mmol) to give322 mg (95%) of AAG154484 as white powder. mp 183-184° C.; ¹H NMR(DMSO-d₆): δ 1.27-1.35 (m, 2H), 1.44-1.67 (m, 6H), 1.87-2.10 (m, 2H),2.31-2.35 (t, J=7.4 Hz, 2H), 2.37-2.44 (m, 4H), 4.30-4.38 (m, 1H), 5.84(s, 1H), 5.95 (s, 2H), 7.07-7.08 (d, J=4 Hz, 1H), 7.77-7.78 (d, J=4 Hz,1H), 8.60-8.62 (d, J=8 Hz, 1H), 10.12 (s, 1H), 10.79 (s, 1H), 12.46 (br,2H). Anal. (C₂₂H₂₇N₅O₆S) C, H, N, S. calcd for C₂₂H₂₇N₅O₆S.1 H₂O; C,52.06; H, 5.64; N, 16.69; S, 6.32; Found: C, 52.39; H, 5.24; N, 13.30;S, 5.98.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-but-1-ynyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154468)

Compound AAG154484 was prepared using the general method described forthe preparation of target compounds, from 6a (50 mg, 0.1 mmol) to give42 mg (95%) of AAG154468 as white powder. mp 196-197° C.; ¹H NMR(DMSO-d₆): δ 1.85-2.11 (m, 2H), 2.31-2.36 (t, J=7.4 Hz, 2H), 2.76 (m,4H), 4.30-4.38 (m, 1H), 5.92 (s, 1H), 5.99 (s, 2H), 7.20-7.21 (d, J=4Hz, 1H), 7.74-7.75 (d, J=4 Hz, 1H), 8.69-8.71 (d, J=8 Hz, 1H), 10.15 (s,1H), 10.86 (s, 1H), 12.47 (br, 2H). HRMS calcd for C₂₀H₁₉N₅O₆S (M⁺),458.1134; found: 458.1155.

(S)-2-({5-[6-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-hex-1-ynyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154479)

Compound AAG154479 was prepared using the general method described forthe preparation of target compounds, from 6c (50 mg, 0.09 mmol) to give43 mg (95%) of AAG154479 as white powder. mp 197-198° C.; ¹H NMR(DMSO-d₆): δ 1.44-1.67 (m, 4H), 1.87-2.10 (m, 2H), 2.23-2.45 (m, 4H),4.30-4.38 (m, 1H), 5.87 (s, 1H), 5.95 (s, 2H), 7.21-7.22 (d, J=4 Hz,1H), 7.73-7.74 (d, J=4 Hz, 1H), 8.66-8.68 (d, J=8 Hz, 1H), 10.12 (s,1H), 10.81 (s, 1H), 12.58 (br, 2H). HRMS calcd for C₂₂H₂₃N₅O₆S (M⁺),486.1447; found: 486.1452.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-but-1-ynyl]-furan-2-carbonyl}-amino)-pentanedioicacid diethyl ester (10)

Compound 10 was prepared using the general method described for thepreparation of 6a-c, from 5a (202 mg, 1 mmol) to give 249 mg (50%) of 10as brown powder. mp 78-79° C.; ¹H NMR (DMSO-d₆): δ 1.14-1.18 (m, 6H),1.92-2.14 (m, 2H), 2.35-2.39 (t, J=7.6 Hz, 2H), 2.77-2.80 (m, 4H),3.99-4.11 (m, 4H), 4.35-4.42 (m, 1H), 5.99 (s, 1H), 6.00 (s, 2H),6.78-6.79 (d, J=3.6 Hz, 1H), 7.14-7.15 (d, J=3.6 Hz, 1H), 8.71-8.73 (d,J=7.6 Hz, 1H), 10.15 (s, 1H), 10.88 (s, 1H).

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-butyl]-furan-2-carbonyl}-amino)-pentanedioicacid diethyl ester (11)

Compound 11 was prepared using the general method described for thepreparation of 7a-c, from 10 (249 mg, 0.5 mmol) to give 231 mg (92%) of11 as yellow powder. mp 71-72° C.; ¹H NMR (DMSO-d₆): δ1.14-1.18 (m, 6H),1.60-1.65 (m, 4H), 1.94-2.10 (m, 2H), 2.36-2.39 (t, J=7.2 Hz, 2H),2.65-2.68 (m, 4H), 4.00-4.11 (m, 4H), 4.34-4.40 (m, 1H), 5.85 (s, 1H),5.95 (s, 2H), 6.25-6.26 (d, J=3.6 Hz, 1H), 7.05-7.06 (d, J=3.6 Hz, 1H),8.43-8.45 (d, J=7.6 Hz, 1H), 10.10 (s, 1H), 10.80 (s, 1H).

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-butyl]-furan-2-carbonyl}-amino)-pentanedioicacid (AAG154489)

Compound AAG154489 was prepared using the general method described forthe preparation of target compounds, from 11 (231 mg, 0.46 mmol) to give194 mg (95%) of AAG154489 as white powder. mp 174-175° C.; ¹H NMR(DMSO-d₆): δ 1.62 (m, 4H), 1.87-2.10 (m, 2H), 2.28-2.32 (t, J=8 Hz, 2H),2.65-2.68 (m, 4H), 4.29-4.43 (m, 1H), 5.86 (s, 1H), 5.95 (s, 2H),6.25-6.26 (d, J=3.6 Hz, 1H), 7.04-7.05 (d, J=3.6 Hz, 1H), 8.29-8.31 (d,J=8 Hz, 1H), 10.12 (s, 1H), 10.80 (s, 1H), 12.47 (br, 2H). Anal.(C₂₀H₂₃N₅O₇) C, H, N. calcd. for C₂₀H₂₃N₅O₇.1.25H₂O; C, 51.33; H, 5.49;N, 14.62; Found: C, 51.34; H, 5.36; N, 14.62.

(S)-2-({5-[4-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-but-1-ynyl]-furan-2-carbonyl}-amino)-pentanedioicacid (AAG154485)

Compound AAG154485 was prepared using the general method described forthe preparation of target compounds, from 10 (50 mg, 0.1 mmol) to give42 mg (95%) of AAG154485 as white powder. mp 193-194° C.; ¹H NMR(DMSO-d₆): δ 1.88-2.11 (m, 2H), 2.28-2.32 (t, J=8 Hz, 2H), 2.78-2.80 (m,4H), 4.30-4.38 (m, 1H), 5.98 (s, 1H), 5.99 (s, 2H), 6.78-6.79 (d, J=3.6Hz, 1H), 7.14-7.15 (d, J=3.6 Hz, 1H), 8.57-8.59 (d, J=8 Hz, 1H), 10.16(s, 1H), 10.88 (s, 1H), 12.45 (br, 2H). HRMS calcd for C₂₀H₁₉N₅O₇(M⁺),441.1284; found: 441.1286.

General Procedure for the Synthesis of Compound 9 and 13

To a solution of 8 or 12 (10 mmol) in anhydrous DMF (20 mL) was addedN-methylmorpholine (1.3 mL, 18 mmol) and2-chloro-4,6-dimethoxy-1,3,5-triazine (2.16 g, 18 mmol). The resultingmixture was stirred at room temperature for 2 h. To this mixture wasadded N-methylmorpholine (1.3 mL, 18 mmol) and L-glutamate diethyl esterhydrochloride (3.6 g, 15 mmol). The reaction mixture was stirred for anadditional 4 h at room temperature and then evaporated to dryness underreduced pressure. The residue was dissolved in the minimum amount ofCH₃Cl/MeOH (4:1) and chromatographed on a silica gel column (2×15 cm)and with 5% EtOAc in hexane as the eluent. Fractions that showed thedesired spot (TLC) were pooled and the solvent evaporated to dryness toafford 9 and 13 in 72% yield.

(S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethylester (9)

Compound 9 was prepared using the general method described for thepreparation of compound 9 and 13, from 8 (2.07 g, 10 mmol) to give 2.82g (72%) of 9 as yellow oil. ¹H NMR (DMSO-d₆): δ 1.14-1.19 (m,6H),1.91-2.12 (m, 2H), 2.39-2.42 (t, J=5.6 Hz, 2H), 4.01-4.12 (m, 4H),4.35-4.39 (m, 1H), 7.30-7.31 (d, J=3.2 Hz, 1H), 7.69-7.70 (d, J=3.2 Hz,1H), 8.81-8.82 (d, J=6 Hz, 1H).

(S)-2-[(5-bromo-furan-2-carbonyl)-amino]-pentanedioic acid diethyl ester(13)

Compound 13 was prepared using the general method described for thepreparation of compound 9 and 13, from 12 (1.91 g, 10 mmol) to give 2.71g (72%) of 13 as yellow oil. ¹H NMR (DMSO-d₆): δ 1.13-1.19 (m,6H),1.88-2.11 (m, 2H), 2.37-2.41 (t, J=7.2 Hz, 2H), 4.00-4.12 (m, 4H),4.36-4.41 (m, 1H), 6.77-6.78 (d, J=3.6 Hz, 1H), 7.20-7.21 (d, J=3.6 Hz,1H), 8.72-8.74 (d, J=7.6 Hz, 1H).

Synthesis and Experimental for Compound AAG154544

Chemistry

Target compound AAG154544 was synthesized as shown in Scheme 5.Palladium-catalyzed Sonogashira coupling of5-bromo-thiophene-2-carboxylic acid methyl ester with but-3-yn-1-ol 1(Scheme 1) afforded thiophenebutynyl alcohol 2 (70%), which wascatalytically hydrogenated to give the saturated alcohol 3 inquantitative yield. Subsequent oxidation of 3 using Jones' reagentafforded the carboxylic acid 4 (47%), which was converted to the acidchloride 5 and immediately reacted with diazomethane followed by 48% HBrto give the desired α-bromomethylketone 7. Condensation of2,4-diamino-6-hydroxypyrimidine with 7 at room temperature for 3 daysafforded the 6-substituted pyrrolo[2,3-d]pyrimidines 8 (38%). Hydrolysisof 8 afforded the corresponding free acid 9 (89%). Subsequent couplingwith L-glutamate diethyl ester using2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agent affordedthe diesters 10. Final saponification of the diesters gave the desiredcompound AAG154544.

Experimental Section

All evaporations were carried out in vacuo with a rotary evaporator.Analytical samples were dried in a CHEM-DRY vacuum (0.2 mm Hg) dryingoven over P₂O₅. Melting points were determined on a MELTEMP II meltingpoint apparatus with FLUKE 51 K/J electronic thermometer and areuncorrected. NMR spectra for proton (¹H) were recorded on a BrukerWH-300 (300 MHz) spectrometer. The chemical shift values are expressedin ppm (parts per million) relative to tetramethylsilane as internalstandard; s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,br=broad singlet. The relative integrals of peak areas agreed with thoseexpected for the assigned structures. Thin-layer chromatography (TLC)was performed on PE SIL G/UV silica gel plates with fluorescentindicator, and the spots were visualized under 254 and 365 nmillumination. Proportions of solvents used for TLC are by volume. Columnchromatography was performed on 230-400 mesh silica gel purchased fromFisher, Somerville, N.J. Elemental analyses were performed by AtlanticMicrolab, Inc., Norcross, Ga. Element compositions are within ±0.4% ofthe calculated values. Fractional moles of water or organic solventsfrequently found in some analytical samples of antifolates were notprevented despite 24-48 h of drying in vacuo and were confirmed wherepossible by their presence in the ¹H NMR spectra. High resolution massspectrometry (HRMS) was performed on a Waters Q-TOF (API-US) byDepartment of Chemistry, University of Pittsburgh, Pittsburgh, Pa. Allsolvents and chemicals were purchased from Aldrich Chemical Co. andFisher Scientific and were used as received.

5-(4-Hydroxy-but-1-ynyl)-thiophene-2-carboxylic acid methyl ester (2)

To a 20-mL vial for microwave reaction, were added a mixture ofpalladium chloride (57 mg, 0.32 mmol), triphenylphosphine (104 mg, 0.32mmol), copper iodide (243 mg, 1.28 mmol), triethylamine (8.08 g, 80mmol), 5-bromo-thiophene-2-carboxylic acid methyl ester (1.77 g, 8 mmol)and anhydrous acetonitrile (10 mL). To the stirred mixture, were addedcopper iodide (243 mg, 1.28 mmol) and but-3-yn-1-ol, 1 (588 mg, 8.4mmol), and the vial was sealed and put into the microwave reactor at100° C. for 10 min. After evaporation of solvent under reduced pressure,MeOH (20 mL) was added followed by silica gel (5 g). The resulting plugwas loaded on to a silica gel column (3.5×12 cm) and eluted with hexanefollowed by 20% EtOAc in hexane. Fractions with an R_(f)=0.42(hexane/EtOAc 4:1) were pooled and evaporated to afford 1.17 g of 2 asyellow oil. ¹H NMR (DMSO-d₆): δ 2.59-2.63 (t, J=6.4 Hz, 2H), 3.56-3.59(t, J=6.4 Hz, 2H), 3.81 (s, 3H), 4.95-4.98 (t, J=5.6 Hz, 1H), 7.27-7.28(d, J=4.0 Hz, 1H), 7.69-7.70 (d, J=4.0 Hz, 1H).

5-(4-Hydroxy-butyl)-thiophene-2-carboxylic acid methyl ester (3)

To a Parr flask were added 2 (1.17 g, 5.6 mmol), 10% palladium onactivated carbon (600 mg), and MeOH (100 mL). Hydrogenation was carriedout at 55 psi of H₂ for 4 h. The reaction mixture was filtered throughCelite, washed with MeOH (100 mL) and concentrated under reducedpressure to give 1.14 g of 3 as yellow oil. ¹H NMR (DMSO-d₆): δ1.41-1.48 (m, 2H), 1.61-1.68 (m, 2H), 2.81-2.85 (t, J=7.2 Hz, 2H),3.37-3.42 (m, 2H), 3.77 (s, 3H), 4.40-4.43 (t, J=5.2 Hz, 1H), 6.94-6.95(d, J=3.6 Hz, 1H), 7.63-7.64 (d, J=3.6 Hz, 1H).

5-(3-Carboxy-propyl)-thiophene-2-carboxylic acid methyl ester (4)

A solution of 3 (1.14 g, 5.3 mmol) in acetone (15 mL) was added dropwiseto a cold solution (ice bath) of CrO₃ (3 g, 30 mmol) in sulfuric acid(23 mL) and water (67 mL). After the addition, the resulting solutionwas stirred in an ice bath for an additional 2 h and the solution wasallowed to warm to room temperature overnight. TLC indicated thedisappearance of the starting alcohol and the formation of one majorspot at R_(f)=0.35 (hexane/EtOAc 2:1). The solution was extracted with5×30 mL of ethyl ether and dried over Na₂SO₄. After evaporation of thesolvent under reduced pressure, the resulting residue was flashchromatographed through silica gel column (3.5 ×15 cm) usinghexane/EtOAc (2:1) as eluent. The desired fraction (TLC) was collectedand the solvent was evaporated under reduced pressure to afford 570 mgof 4 as colorless oil. ¹H NMR (DMSO-d₆): δ 1.79-1.87 (m, 2H), 2.24-2.27(t, J=7.2 Hz, 2H), 2.82-2.86 (t, J=7.2 Hz, 2H), 3.77 (s, 3H), 6.95-6.96(d, J=3.6 Hz, 1H), 7.63-7.64 (d, J=3.6 Hz, 1H), 12.17 (br, 1H). HRMScalcd for C₁₀H₁₂O₄S (M⁺), 228.0456; found: 228.0458.

5-(5-Bromo-4-oxo-pentyl)-thiophene-2-carboxylic acid methyl ester (7)

To 4 (570 mg, 2.5 mmol) in a 100 mL flask were added oxalyl chloride(1.9 g, 15 mmol) and anhydrous CH₂Cl₂ (20 mL). The resulting solutionwas refluxed for 1 h and then cooled to room temperature. Afterevaporating the solvent under reduced pressure, the residue 5 wasdissolved in 20 mL of Et₂O. The resulting solution was added dropwise toan ice-cooled diazomethane (generated in situ from 10 g of diazald byusing Aldrich Mini Diazald Apparatus) in an ice bath over 10 min. Theresulting mixture was allowed to stand for 30 min and then stirred foran additional 1 h. To this solution was added 48% HBr (20 mL). Theresulting mixture was refluxed for 1.5 h. After cooling to roomtemperature, the organic layer was separated and the aqueous layerextracted with Et₂O (50 mL×2). The combined organic layer and Et₂Oextract was washed with two portions of 10% Na₂CO₃ solution and driedover Na₂SO₄. Evaporation of the solvent afforded 7 as colorlesscrystals. ¹H NMR (CDCl₃-d): δ 1.99-2.07 (m, 2H), 2.71-2.75 (t, J=7.2 Hz,2H), 2.87-2.91 (t, J=7.2 Hz, 2H), 3.87 (s, 3H), 3.88 (s, 2H), 6.81-6.82(d, J=3.6 Hz, 1H), 7.64-7.65 (d, J=3.6 Hz, 1H). HRMS calcd forC₁₁H₁₃BrO₃S (M⁺), 303.9769; found: 303.9759.

5-[3-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-propyl]-thiophene-2-carboxylicacid methyl ester (8)

To a suspension of 2,6-diaminopyrimidin-4-one (315 mg, 2.5 mmol) inanhydrous DMF (15 mL) was added 7 (about 2.4 mmol). The resultingmixture was stirred under N₂ at room terperature for 3 days. Afterevaporation of solvent under reduced pressure, MeOH (20 mL) was addedfollowed by silica gel (1.5 g). The resulting plug was loaded on to asilica gel column (3.5×12 cm) and eluted with CHCl₃ followed by 3% MeOHin CHCl₃ and then 5% MeOH in CHCl₃. Fractions with an R_(f)=0.56(MeOH/CHCl₃ 1:5) were pooled and evaporated to afford 300 mg of 8 aswhite powder in 38% yield. ¹H NMR (DMSO-d₆): δ 1.89-1.97 (m, 2H),2.49-2.54 (t, J=7.2 Hz, 2H), 2.82-2.85 (t, J=7.2 Hz, 2H), 3.78 (s, 3H),5.89 (s, 1H), 5.96 (s, 2H), 6.97-6.98 (d, J=3.6 Hz, 1H), 7.64-7.65 (d,J=3.6 Hz, 1H), 10.13 (s, 1H), 10.82 (s, 1H).

5-[3-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-propyl]-thiophene-2-carboxylicacid (9)

To a solution of 8 (300 mg, 0.9 mmol) in MeOH (10 mL) was added 1 N NaOH(10 mL) and the mixture was stirred under N₂ at room temperature for 16h. TLC showed the disappearance of the starting material (R_(f)=0.56)and one major spot at the origin (MeOH/CHCl₃ 1:5). The reaction mixturewas evaporated to dryness under reduced pressure. The residue wasdissolved in water (10 mL), the resulting solution was cooled in an icebath, and the pH was adjusted to 3-4 with dropwise addition of 1 N HCl.The resulting suspension was frozen in a dry ice-acetone bath, thawed to4-5° C. in the refrigerator, and filtered. The residue was washed with asmall amount of cold water and dried in vacuum using P₂O₅ to afford 254mg of 9 as white powder. ¹H NMR (DMSO-d₆): δ 1.89-1.96 (m, 2H),2.49-2.55 (t, J=7.2 Hz, 2H), 2.80-2.84 (t, J=7.2 Hz, 2H), 5.88 (s, 1H),5.98 (s, 2H), 6.92-6.93 (d, J=3.6 Hz, 1H), 7.55-7.56 (d, J=3.6 Hz, 1H),10.14 (s, 1H), 10.83 (s, 1H) 12.86 (br, 1H).

(S)-2-({5-[3-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-propyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid diethyl ester (10)

To a solution of 9 (254 mg, 0.8 mmol) in anhydrous DMF (10 mL) was addedN-methylmorpholine (145 mg, 1.44 mmol) and2-chloro-4,6-dimethoxy-1,3,5-triazine (253 g, 1.44 mmol). The resultingmixture was stirred at room temperature for 2 h. To this mixture wereadded N-methylmorpholine (145 mg, 1.44 mmol) and L-glutamate diethylester hydrochloride (290 mg, 1.2 mmol). The reaction mixture was stirredfor an additional 4 h at room temperature and then evaporated to drynessunder reduced pressure. The residue was dissolved in the minimum amountof CHCl₃/MeOH (4:1) and chromatographed on a silica gel column (2×15 cm)and with 5% CHCl₃ in MeOH as the eluent. Fractions that showed thedesired spot (TLC) were pooled and the solvent evaporated to dryness toafford 252 mg of 10 as yellow powder in 63% yield. ¹H NMR (DMSO-d₆): δ1.14-1.21 (m, 6H), 1.81-2.05 (m, 4H), 2.32-2.39 (t, J=7.6 Hz, 2H),2.49-2.52 (t, J=7.2 Hz, 2H), 2.78-2.81 (t, J=7.2 Hz, 2H), 4.02-4.07 (m,4H), 4.30-4.35 (m, 1H), 5.88 (s, 1H), 5.94 (s, 2H), 6.89-6.90 (d, J=3.6Hz, 1H), 7.68-7.69 (d, J=3.6 Hz, 1H), 8.61-8.63 (d, J=8 Hz, 1H), 10.71(s, 1H), 11.19 (s, 1H).

(S)-2-({5-[3-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-propyl]-thiophene-2-carbonyl}-amino)-pentanedioicacid (AAG154544)

To a solution of 10 (252 mg, 0.5 mmol) in MeOH (10 mL) was added 1 NNaOH (10 mL) and the mixture was stirred under N₂ at room temperaturefor 16 h. TLC showed the disappearance of the starting material(R_(f)=0.48) and one major spot at the origin (MeOH/CHCl₃ 1:5). Thereaction mixture was evaporated to dryness under reduced pressure. Theresidue was dissolved in water (10 mL), the resulting solution wascooled in an ice bath, and the pH was adjusted to 3-4 with dropwiseaddition of 1 N HCl. The resulting suspension was frozen in a dryice-acetone bath, thawed to 4-5° C. in the refrigerator, and filtered.The residue was washed with a small amount of cold water and dried invacuum using P₂O₅ to afford 212 mg (95%) of AAG154544 as white powder.mp 175-176° C.; ¹H NMR (DMSO-d₆): δ 1.88-2.10 (m, 4H), 2.31-2.34 (t,J=7.6 Hz, 2H), 2.49-2.54 (t, J=7.2 Hz, 2H), 2.78-2.81 (t, J=7.2 Hz, 2H),4.30-4.35 (m, 1H), 5.88 (s, 1H), 5.97 (s, 2H), 6.89-6.90 (d, J=3.6 Hz,1H), 7.68-7.69 (d, J=3.6 Hz, 1H), 8.50-8.52 (d, J=8 Hz, 1H), 10.13 (s,1H), 10.82 (s, 1H) 12.42 (br, 2H). Anal. (C₁₉H₂₁N₅O₆S) C, H, N, S. calcdfor C₁₉H₂₁N₅O₆S.1 H₂O; C, 49.02; H, 4.98; N, 15.05; S, 6.89; Found: C,49.22; H, 4.80; N, 15.03; S, 6.97.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims

1. A compound of Formula I:

wherein R₁ is one of (a) a hydrogen (H)), (b) an OH, (c) a CH₃, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms, and tautomers of said (b) and said (d); R₂ is one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A is CR′R″, whereinR′ and R″ are the same or different and are either a H or an alkyl grouphaving from 1 to 6 carbon atoms; wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof, and R₃ is oneof (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl, (d) difluoromethyl,(e) monofluoromethyl, (f) methyl ketone, (g) trifluoromethyl ketone, (h)difluoromethyl ketone, (i) monofluoromethyl ketone, (j) formyl, (k)methyl alcohol, (l) methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, and the sum total of integers y andz is equal to zero, 5, 6, or
 7. 2. The compound of claim 1 comprisingwherein said side chain attachment is at carbon atom position 6, andfurther comprising wherein the carbon atom at position 5, independentlyhas attached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.
 3. The compound of claim 1 comprising wherein saidside chain attachment is at carbon atom position 5, and furthercomprising wherein the carbon atom at position 6, independently hasattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.
 4. The compound of claim 1 comprising wherein saidheterocycloalkyl-carbonyl-L-glutamate group is selected from the groupconsisting of a dihydrothiophene-carbonyl-L-glutamate group, atetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof.
 5. Thecompound of claim 1 wherein said side chain has one or more carbon tocarbon double or triple bonds between the carbon atoms of (C)_(y) and(C)_(z).
 6. The compound of claim 1 wherein saidheterocycloaryl-carbonyl-L-glutamate group is selected from the groupconsisting of a thiophene-carbonyl-L-glutamate group, afuran-carbonyl-L-glutamate group, a pyrrole-carbonyl-L-glutamate group,and a pyridine-carbonyl-L-glutamate group.
 7. The compound of claim 1wherein said side chain of Formula I has one or more double bondscomprising E-isomers and Z-isomers.
 8. A pharmaceutically acceptablesalt of the compound of Formula I:

wherein R₁ is one of (a) a hydrogen (H)), (b) an OH, (c) a CH₃, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms, and tautomers of said (b) and said (d); R₂ is one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A is CR′R″, whereinR′ and R″ are the same or different and are either a H or an alkyl grouphaving from 1 to 6 carbon atoms; wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof, and R₃ is oneof (a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl, (d) difluoromethyl,(e) monofluoromethyl, (f) methyl ketone, (g) trifluoromethyl ketone, (h)difluoromethyl ketone, (i) monofluoromethyl ketone, (j) formyl, (k)methyl alcohol, (l) methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is a aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, and wherein the sum total ofintegers y and z is equal to zero, 5, 6, or
 7. 9. A pharmaceuticalcomposition of a therapeutically effective amount of a compound ofFormula I, or a pharmaceutically acceptable salt of Formula I:

wherein R₁ is one of (a) a hydrogen (H)), (b) an OH, (c) a CH₃, and (d)NHR wherein R is either a H or an alkyl group having from 1 to 6 carbonatoms, and tautomers of said (b) and said (d); R₂ is one of (a) ahydrogen (H), (b) a CH₃, (c) an OH, and (d) NHR wherein R is either a Hor an alkyl group having from 1 to 6 carbon atoms; A is CR′R″, whereinR′ and R″ are the same or different and are either a H or an alkyl grouphaving from 1 to 6 carbon atoms; wherein the bond at position 5-6 mayeither be a single or a double bond; wherein the five membered ring hasa side chain attached at positions 5, 6 or 7, and optionally includeswherein the carbon atoms at positions 5 and 6, independently, haveattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms 5 and 6 is a single bond or one hydrogen atom if the bondbetween carbon atoms 5 and 6 is a double bond, or (b) an alkyl grouphaving from one to six carbon atoms and a hydrogen atom if the bondbetween carbon atoms at positions 5 and 6 is a single bond or an alkylgroup having from one to six carbon atoms if the bond between carbonatoms 5 and 6 is a double bond, and combinations thereof; R₃ is one of(a) a hydrogen (H), (b) CH₃, (c) trifluoromethyl, (d) difluoromethyl,(e) monofluoromethyl, (f) methyl ketone, (g) trifluoromethyl ketone, (h)difluoromethyl ketone, (i) monofluoromethyl ketone, (j) formyl, (k)methyl alcohol, (l) methylamine, or (m) a bond; X is either aheterocycloalkyl-carbonyl-L-glutamate group, aheterocycloaryl-carbonyl-L-glutamate group, or a hydrogen (H), andwherein X is a hydrogen then R₄ is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group, and wherein X is aheterocycloalkyl-carbonyl-L-glutamate group or aheterocycloaryl-carbonyl-L-glutamate group then R₄ is a hydrogen or abond; wherein R₅ is the same as R₃ except that R₅ is not a bond; y is aninteger ranging from zero up to and including 6; z is an integer rangingfrom zero up to and including seven, and the sum total of integers y andz is equal to zero, 5, 6, or
 7. 10. The pharmaceutical composition ofclaim 9 wherein said side chain attachment is at carbon atom position 6,and further wherein the carbon atom at position 5, independently hasattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.
 11. The pharmaceutical composition of claim 9wherein said side chain attachment is at carbon atom position 5, andfurther wherein the carbon atom at position 6, independently hasattached thereto either (a) two hydrogen atoms if the bond betweencarbon atoms at positions 5 and 6 is a single bond or one hydrogen atomif the bond between carbon atoms at positions 5 and 6 is a double bond,or (b) an alkyl group having from one to six carbon atoms if the bondbetween carbon atoms of positions 5 and 6 is a double bond or an alkylgroup having from one to six carbon atoms and a hydrogen atom if thebond between carbon atoms at positions 5 and 6 is a single bond, andcombinations thereof.
 12. The pharmaceutical composition of claim 9wherein said heterocycloalkyl-carbonyl-L-glutamate group is selectedfrom the group consisting of a dihydrothiophene-carbonyl-L-glutamategroup, a tetrahydrothiophene-carbonyl-L-glutamate group, adihydrofuran-carbonyl-L-glutamate group, atetrahydrofuran-carbonyl-L-glutamate group, adihydropyrrole-carbonyl-L-glutamate group, atetrahydropyrrole-carbonyl-L-glutamate group, amonohydropyridyl-carbonyl-L-glutamate group, adihydropyridyl-carbonyl-L-glutamate group, and apiperidyl-carbonyl-L-glutamate group, and stereoisomers thereof.
 13. Thepharmaceutical composition of claim 9 wherein said side chain has one ormore carbon to carbon double or triple bonds between the carbon atoms of(C)_(y) and (C).
 14. The pharmaceutical composition of claim 9 whereinsaid heterocycloaryl-carbonyl-L-glutamate group is selected from thegroup consisting of a thiophene-carbonyl-L-glutamate group, afuran-carbonyl-L-glutamate group, a pyrrole-carbonyl-L-glutamate group,and a pyridine-carbonyl-L-glutamate group.
 15. The pharmaceuticalcomposition of claim 9 wherein said side chain of Formula I has one ormore double bonds comprising E-isomers and Z-isomers.
 16. A compound ofFormula II:

wherein R₁ is one of a hydrogen (H) or an alkyl group having from 1 to 6carbon atoms; R₂ is one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and(d) NHR wherein R is either a H or an alkyl grouphaAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAvingfrom 1 to 6 carbon atoms; A is one of (a) CR′R″, (b) NR′, wherein R′ andR″ are the same or different and are either a H or an alkyl group havingfrom 1 to 6 carbon atoms, and (c) an oxygen (O); wherein the fivemembered ring has a side chain attached at position 6, and optionallyincludes wherein the carbon atom at position 5, has attached theretoeither (a) one hydrogen atom, or (b) an alkyl group having from one tosix carbon atoms, and R₃ is one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; B is one of (a) a sulfur (S) atom, (b) anoxygen (O) atom, or (c) a nitrogen (N) atom; and y is an integer rangingfrom zero up to and including
 7. 17. The compound of claim 16,consisting of said side chain having one or more carbon to carbon doubleor triple bonds between the carbon atoms of (C)_(y 1-7).
 18. Thecompound of claim 16 wherein said side chain of Formula II has or one ormore double bonds comprising E-isomers and Z-isomers.
 19. Apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula II or a pharmaceutically acceptable salt of acompound of Formula II:

wherein R₁ is one of a hydrogen (H) or an alkyl group having from 1 to 6carbon atoms; R₂ is one of (a) a hydrogen (H), (b) a CH₃, (c) an OH, and(d) NHR wherein R is either a H or an alkyl group having from 1 to 6carbon atoms; A is one of (a) CR′R″, (b) NR′, wherein R′ and R″ are thesame or different and are either a H or an alkyl group having from 1 to6 carbon atoms, and (c) an oxygen (O); wherein the five membered ringhas a side chain attached at position 6, and optionally includes whereinthe carbon atom at position 5 has attached thereto either (a) onehydrogen atom, or (b) an alkyl group having from one to six carbonatoms, and R₃ is one of (a) a hydrogen (H), (b) CH₃, (c)trifluoromethyl, (d) difluoromethyl, (e) monofluoromethyl, (f) methylketone, (g) trifluoromethyl ketone, (h) difluoromethyl ketone, (i)monofluoromethyl ketone, (j) formyl, (k) methyl alcohol, (l)methylamine, or (m) a bond; B is one of (a) a sulfur (S) atom, (b) anoxygen (O), or (c) a nitrogen (N) atom; and y is an integer ranging fromzero up to and including
 7. 20. The compound of Formula II of claim 19wherein said side chain has one or more carbon to carbon double ortriple bonds between the carbon atoms of (C)_(y 1-7).